Novel Goodpasture Antigen-Binding Protein Isoforms and Protein Misfolded-Mediated Disorders

ABSTRACT

The present invention provides novel isoforms of the Goodpasture antigen binding protein (GPBP), and related reagents, and also provides methods for isolating and detecting such novel GPBP isoforms. The invention further provides methods identifying compounds to treat one or more of an autoimmune condition and a protein deposit-mediated disorder, as well as novel compounds and methods for treating such conditions and/or disorders.

CROSS REFERENCE

This application claims priority to U.S. Provisional Patent ApplicationSer. Nos. 60/445,043 filed Feb. 5, 2003; 60/445,003 filed Feb. 5, 2003;and 60/445,004 filed Feb. 5, 2003, which are herewith incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

The conformation of the non-collagenous (NC1) domain of the α3 chain ofthe basement membrane collagen IV [α3(IV)NC1] depends in part onphosphorylation. Goodpasture Antigen Binding Protein (GPBP) (WO00/50607; WO 02/061430) is a novel non-conventional protein kinase thatcatalyzes the conformational isomerization of the α3(IV)NC1 domainduring its supramolecular assembly, resulting in the production andstabilization of multiple α3(IV)NC1 conformers in basement membranes.Elevated levels of GPBP have been associated with the production ofnon-tolerized α3(IV)NC1 conformers, which conduct the autoimmuneresponse mediating Goodpasture (“GP”) disease. In GP patients,autoantibodies against the non-collagenous C-terminal domain (NC1) ofthe type IV collagen a3 chain (“Goodpasture antigen” or “GP antigen”)cause a rapidly progressive glomerulonephritis and often lunghemorrhage, the two cardinal clinical manifestations of the GP syndrome.

The identification of GPBP provided methods for identification ofcompounds for the treatment of autoimmune disorders, cancer, andaberrant apoptosis, and also provided potential therapeutics for thesedisorders. Thus, the identification of novel GPBP isoforms would beadvantageous in at least these fields.

SUMMARY OF THE INVENTION

The present invention provides novel isoforms of the Goodpasture antigenbinding protein (GPBP), and related reagents, and also provides methodsfor isolating and detecting such novel GPBP isoforms. The inventionfurther provides methods identifying compounds to treat one or more ofan autoimmune condition and a protein deposit-mediated disorder, as wellas novel compounds and methods for treating such conditions and/ordisorders.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. 91-kDa GPBP represents a non-canonical translation product ofthe mRNA. The cDNAs present in pcDNΔ3 (1), pc-n4′(2) or pc-n4′-Met_(mut)(3) were expressed in a cell-free system (in vitro) or in human 293cells (ex vivo) and similar amounts of the corresponding mixtures orextracts were analyzed by fluorography (in vitro) or by Western blotusing Mab6 antibodies (ex vivo), respectively. Unless otherwiseindicated with numbers and bars we indicate in this and the followingFigures the size in kDa and position of rainbow molecular weight markersfrom Amersham Bioscience.

FIG. 2. The 91-kDa GPBP represent a non-canonical translation of the ORFexisting in 5′UTR of the mRNA. In A, the ORF of the 5′-UTR of human GPBPmRNA is written in capitals and one-letter code. The 5′ end and thetranslation direction of the indicated pcDNΔ3-based constructs aremarked with bent arrows. The sequence of the synthetic peptide(GPBPpep2) is highlighted. In B, the cDNAs in the indicated constructswere expressed in a cell-free system (in vitro) or in human 293 cells(ex vivo) and analyzed as in FIG. 1.

FIG. 3. In the cell the non-canonical 91-kDa GPBP isoform is moreabundant than canonical 77-kDa. Lysates from pc-n4′ (1) or fromnon-transfected (2,3) 293 cells were analyzed by Western blot with Mab6(1,2) or with Mab6 and GPBPpep1 (3).

FIG. 4. Cellular 91- and 120-kDa GPBP-related polypeptides aretranslation products of GPBP mRNA. Similar amounts (50 μg) of lysatesfrom non-transfected 293 cells (1) or from 293 cells transfected with aplasmid encoding for SiGFP (2), SiGPBP (3), SiGPBP/Δ26-1 (4)SiGPBP/Δ26-2 (5), SiGPBP/Δ26-3 (6), SiGPBP/Δ26-4 (7) were analyzed byWestern blot using the indicated antibodies.

FIG. 5. Localization of GPBP by subcellular fractioning of rathepatocytes. Similar amounts (˜50 μg) of homogenate (1), cytosol (2),microsomes (3), mitochondria (4) and lysosomes (5) isolated from ratliver were analyzed by Western blot using Mab6 antibodies. Parallelstudies performed in the absence of Mab6 revealed no immunoreactivepolypeptides in any of the fractions analyzed.

FIG. 6. Identification of 91-kDa GPBP isoform in rat liver lysosomes andevidence for processing to 44-47-kDa isoforms. In A, similar amounts(˜50 μg) of lysosomal fractions from liver of untreated (C) orleupeptin-treated (L) rats were analyzed by Western blot using Mab6antibodies. In B, lysosomal fractions as in A were further fractionedand whole (W), soluble (S) or non-soluble (M) fractions were similarlyanalyzed.

FIG. 7. Lysosomal proteolysis of in vitro expressed GPBP generatespolypeptides of similar molecular mass than endogenous GPBP-relatedpolypeptides. The cDNA in pc-n4′ was expressed in a cell-free system andsimilar amounts of the mixtures were incubated in the absence oflysosomal extract for 20 min (1) or in the presence of lysosomal extractfor 5 (2) or 20 (3) min and analyzed by SDS-PAGE and fluorography.

FIG. 8. Phosphate transfer activity in isolated rat liver lysosomes. InA, entire (1,2,3) or broken (4,5,6) rat liver lysosomes were incubatedfor 0 (1,4), 10 (2,5) or 20 (3,6) min with a phosphorylation mixturecontaining [γ³²P]ATP and further analyzed by SDS-PAGE andautoradiography. In B, entire lysosomes from liver of untreated(Control) or leupeptin-treated (Leupeptin) rats were similarly incubatedfor 0 (1), 15 (2), 30 (3) or 60 (4) min and further analyzed by Westernblot using Mab6 (Western) and autoradiography (³²P). Here and in thefollowing Figures the autoradiographic study was performed first toavoid labeling leakage during Western blot processing. With numbers andbars we indicate the size in kDa and position of Mab6 reactivepolypeptides on either study. The arrows denote the autoradiographicbands whose intensity increased during time of incubation.

FIG. 9. Conformational diversification of the α3(IV)NC1 domain occurs atthe endosomal-lysosomal compartment and depends on GPBP. In A, 293 cellsexpressing recombinant α3(IV)NC1 domain were treated with 20 mM NH₄Cland/or 100 μM leupeptin. Similar amounts of serum-free media wereanalyzed by SDS-PAGE under reducing (R) or non-reducing (NR) conditionsand Western-blot using α3(IV)NC1-specific antibodies (Mab175). In B,similar amounts of recombinant GPBP or α3(IV)NC1-expressing cells wereincubated or cultured respectively in the absence (Con) or in thepresence of the indicated GPBP modulator. Phosphorylation mixtures wereanalyzed as in FIG. 8 (³²P) using Mab14 in the Western blot staining todetermine that were not differences in the amount of recombinant proteinamong lanes (not shown). Culture media were analyzed as in A (Western).

FIG. 10. GPBP interacts and phosphorylates PrP^(C). In A, cellularextracts of cultured rat cerebellar neurons were analyzed by SDS-PAGEand Western blot using PrP (C-20) antibodies (α-PrP^(C)) or by farWestern blot using recombinant GPBP and Mab14 (far Western). In B, 1 μgof bovine recombinant PrP (Prionics), human recombinant α3(IV)NC1 (C+)or horse heart cytochrome c from Sigma (C−) were analyzed by SDS-PAGEand Coomassie blue stained (Coomassie) or by far Western blot as in A(GPBP+α-GPBP). In C, 100 ng of human recombinant GPBP (1), same amountof GPBP with 1 μg of bovine recombinant PrP (2) or the same amount ofbovine recombinant PrP (3) were separately subjected to in vitrophosphorylation and the corresponding mixtures analyzed by Western blotusing PrP specific antibodies in A (not shown) and autoradiography(³²P). With an arrow we note the position of recombinant PrP.

FIG. 11. PrP and GPBP interact in cells lysates. Cultured 293 cells weretransfected with pc-DNΔ3 and pc-PrP (1), pc-DNΔ3 and pc-PrP^(E168R) (2),pc-Flag-n4′ and pc-PrP (3) or with pc-Flag-n4′ and pc-PrP^(E168R ()4),lysed and subjected to anti-FLAG immunoprecipitation. Lysates andimmunoprecipitated (IP) materials were analyzed by Western blot usingthe indicated biotin-labeled antibodies.

FIG. 12. Evidence for GPBP modulators regulating human recombinant PrPconformation in 293 cells. Human 293 cells were transfected with pc-PrP,cultured in the absence (C) or in the presence of DAB-Am-32 (D32) orDAB-Am4 (D4) and further lysed and centrifuged. The correspondingsupernatants (st) and pellets were analyzed by Western blot using 3F4anti-PrP antibodies. Similar results to those obtained with DAB-Am-32were also observed with Q_(2D) (not shown).

FIG. 13. Evidence for GPBP mRNA silencers regulating recombinant PrPconformation in 293 cells. Human 293 cells were transfected with pc-PrPand either SiGFP (C), SiGPBP/Δ26-2 (1) or SiGPBP/Δ26-4 (2) cultured for48 h and further lysed and centrifuged. The corresponding supernatants(st) and pellets were analyzed by Western blot using 3F4 anti-PrPantibodies. Western blot analysis on the cell lysates confirmed thatSiGPBP/Δ26-2 silenced endogenous GPBP more efficiently than SiGPBP/Δ26-4(not shown).

FIG. 14. Evidences for GPBP interacting with Aβ₁₋₄₂. Similar amounts (1μg) of Aβ₁₋₄₂ (1) or GPpep1bov (2) were analyzed by far Western blot asin previous Figures. The presence of similar amounts of each of the twopolypeptides in the Immobilon P membrane was determined either withspecific antibodies reacting with each polypeptide or by Ponceau Sstaining (not shown).

DETAILED DESCRIPTION OF THE INVENTION

All references cited are herein incorporated by reference in theirentirety.

Within this application, unless otherwise stated, the techniquesutilized may be found in any of several well-known references such as:Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, SanDiego, Calif.), “Guide to Protein Purification” in Methods in Enzymology(M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.), Culture of Animal Cells: A Manual of BasicTechnique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.),Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J. Murray,The Humana Press Inc., Clifton, N.J.), and the Ambion 1998 Catalog(Ambion, Austin, Tex.).

As used herein, the term “GPBP” or “GPBP isoform” refers to Goodpastureantigen binding protein, and includes the various alternative GPBPisoforms disclosed herein, including GPBPΔ26 isoforms, and furtherincludes both monomers and oligomers thereof. The various GPBP isoformsdisclosed herein include 91 kDa GPBP, 77 kDa GPBP, 60 kDa GPBP, 44-47kDa GPBP, and 32 kDa GPBP. Human, mouse, and bovine isoforms areprovided herein.

As used herein, the term “GPBPΔ26” refers to Goodpasture antigen bindingprotein deleted for the 26 amino acid sequence shown in SEQ ID NO: 46,and the various alternative GPBP isoforms disclosed herein, and furtherincludes both monomers and oligomers thereof. The various GPBPΔ26isoforms disclosed herein include 91 kDa GPBPΔ26, and 77 kDa GPBPΔ26.Human, mouse, and bovine isoforms are provided herein.

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,reference to a “GPBP isoform” means one or more GPBP isoforms.

As used herein the term “non-canonical” means that the GPBP beingreferred to is not expressed from the methionine initiation codon thatyields 77 kDa GPBP or 77 kDa GPBPΔ26. For the sake of simplicity,recitations of “non-canonical GPBP” include both non-canonical GPBPisoforms and non-canonical GPBPΔ26 isoforms.

As used herein a “protein deposit-mediated disorder” means a diseasemediated by abnormal deposition of a specific protein, including but notlimited to Parkinson's disease, Alzheimer's disease, amyotrophic lateralsclerosis, prion diseases, type II diabetes, and autoimmune disorders.The protein deposit may be amyloid matter or para-amyloid matter.

As used herein an “autoimmune condition” is selected from the groupconsisting of Goodpasture Syndrome, multiple sclerosis, systemic lupuserythematosus, cutaneous lupus erythematosus, pemphigus, pemphigoid andlichen planus.

The amino acid sequence of 77 kDa GPBP and GPBPΔ26 was disclosed in U.S.Pat. No. 6,579,969 Issued Jun. 17, 2003 and corresponding PCTpublication WO 00/50607, published Aug. 31, 2000. GPBP was identifiedtherein as a 71 kDa protein that underwent post-translationalmodification to result in higher molecular weight polypeptides. It wasalso disclosed that the 71 kDa protein began at a methionine residue,but that in the 5′ untranslated region upstream of the coding regionencoding the amino-terminal methionine of the 71 kDa protein, the cDNAclone encoding 71 kDa GPBP contained an open reading frame without aninitiation codon for translation. It was speculated that an mRNA editingprocess inserting a single base pair (U) might generate an operativein-frame start site and an ORF of 754-residues containing an exportsignal immediately downstream of the edited Met.

The present invention demonstrates that, rather than the mRNA editingprocess speculated on in WO 00/50607, the human GPBP mRNA undergoesnon-canonical translation initiation to produce a 91-kDa isoform of GPBP(91 kDa GPBP). The resulting protein product is not the 753 amino acidresidue protein speculated upon in WO 00/50607, but is believed to be aprotein of approximately 727 amino acid residues comprising the aminoacid sequence of SEQ ID NO:6. The corresponding predicted 91 kDa GPBPΔ26amino acid sequence comprises the amino acid sequence of SEQ ID NO:8.The present invention also provides mouse and bovine homologs of thehuman 91 kDa polypeptide: mouse 91 kDa GPBP (SEQ ID NO:94), mouse 91 kDGPBPΔ26 (SEQ ID NO:96), bovine 91 kDa GPBP (SEQ ID NO:98), and bovine 91kDa GPBPΔ26 (SEQ ID NO:100).

For the sake of simplicity, the different isoforms are referred to asbeing the same molecular weight, whether a GPBP isoform or a GPBPΔ26isoform. It will be apparent to one of skill in the art that the GPBPΔ26isoform will contain 26 fewer amino acid residues than the correspondingGPBP isoform, and thus will have a molecular weight approximately 2.6kDa less than the corresponding GPBP isoform.

The present invention further demonstrates that various processed formsof these GPBP isoforms exist, and provides evidence for the dependencyof their subcellular localization on the particular processing eventthat occurs. The invention further provides a series of truncationmutants of the GPBP cDNA that are predicted to encode the primarysequence signals to direct their differential subcellular localizationpatterns. The expression products of these truncation mutants are asfollows (also, see FIG. 2):

Δ102 GPBP SEQ ID NO: 26 Δ102 GPBPΔ26 SEQ ID NO: 28 Δ174 GPBP SEQ ID NO:22 Δ174 GPBPΔ26 SEQ ID NO: 24 Δ246 GPBP SEQ ID NO: 18 Δ246 GPBPΔ26 SEQID NO: 20 Δ315 GPBP SEQ ID NO: 14 Δ315 GPBPΔ26 SEQ ID NO: 16 Δ369 GPBPSEQ ID NO: 10 Δ369 GPBPΔ26 SEQ ID NO: 12

Thus, in one aspect, the present invention provides substantiallypurified polypeptide comprising or consisting of an amino acid sequenceaccording to SEQ ID NO:29, which is the amino acid sequence present inΔ369 GPBP (or Δ369 GPBPΔ26) that is not present in GPBP (or GPBPΔ26).The amino acid sequence of SEQ ID NO:29 is GAGAGLLLGCRAS. In oneembodiment of this aspect, the substantially purified polypeptidescomprise or consist of the amino acid sequence of SEQ ID NO:30, which isthe amino acid sequence present in Δ315 GPBP (or Δ315 GPBPΔ26) that isnot present in GPBP (or GPBPΔ26).

In a further embodiment of this aspect, the substantially purifiedpolypeptides comprise or consist of the amino acid sequence of SEQ IDNO:31, which is the amino acid sequence present in Δ246 GPBP (or Δ246GPBPΔ26) that is not present in GPBP (or GPBPΔ26). In a furtherembodiment of this aspect, the substantially purified polypeptidescomprise or consist of the amino acid sequence of SEQ ID NO:32, which isthe amino acid sequence present in Δ174 GPBP (or Δ174 GPBPΔ26) that isnot present in GPBP (or GPBPΔ26).

In a further embodiment of this aspect, the substantially purifiedpolypeptides comprise or consist of the amino acid sequence of SEQ IDNO:33, which is the amino acid sequence present in Δ102 GPBP (or Δ102GPBPΔ26) that is not present in GPBP (or GPBPΔ26). In a furtherembodiment of this aspect, the substantially purified polypeptidescomprise or consist of the amino acid sequence of SEQ ID NO:34, which isthe predicted amino acid sequence present in 91 kDa GPBP (or 91 kDaGPBPΔ26) that is not present in GPBP (or GPBPΔ26).

In various further embodiments of this aspect of the invention, thesubstantially purified polypeptides comprise or consist of an amino acidsequence selected from the group consisting of SEQ ID NO:6 (predicted 91kDa GPBP), SEQ ID NO:8 (predicted 91 kDa GPBPΔ26), SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:98(bovine 91 kD GPBP homolog), and SEQ ID NO:100 (bovine 91 kD GPBPΔ26homolog).

In a further aspect, the present invention provides substantiallypurified polypeptides comprising or consisting of an amino acid sequenceaccording to SEQ ID NO:101 (GAGAGLLLGCRVS), which is present in mouseand rat GPBP isoforms, and which corresponds to SEQ ID NO:29 from thehuman sequence but differs in a single amino acid residue (underlined).Sequence comparison of potential open reading frames in the mouse,bovine, and rat GPBP mRNA indicates that they encode sequences that areof great similarity to the human GPBP isoforms disclosed herein: atleast 94% identity for the 91 kDa GPBP between human, mouse, and bovinehomolgs and at least 81% identity between human, mouse, rat and bovinehomolgs for the predicted amino acid sequences upstream of canonicalGPBP. Thus, in another embodiment of this aspect, the present inventionprovides substantially purified polypeptides comprising an amino acidsequence that are at least 80% identical to SEQ ID NO:34. Such sequenceidentity is as determined using the BLAST engine for local alignment.The stand-alone executable for blasting two sequences (bl2seq) can beretrieved from the NCBI internet site, and is also disclosed in FEMSMicrobiol Lett. 174:247-250 (1999).

In another embodiment of this aspect, the present invention providessubstantially purified polypeptides comprising or consisting of an aminoacid sequence selected from the group consisting of SEQ ID NO:94 and SEQID NO:96. These polypeptides represent mouse homologs of human 91 kDaGPBP and 91 kDa GPBPΔ26, respectively.

In these various aspects and embodiments, the present invention providesnovel polypeptides that can be used to generate antibodies todistinguish between different GPBP isoforms, and which can also be used,for example, as tools to identify candidate compounds for inhibitingvarious specific types of GPBP isoforms and also to identify candidatecompounds for treating autoimmunity and amyloidosis disorders, asdiscussed in more detail below.

As used herein, the term “substantially purified” means that the proteinhas been separated from its in vivo cellular environments. Thus, theprotein can either be purified from natural sources, or recombinantprotein can be purified from the transfected host cells disclosed above.In a preferred embodiment, the proteins are produced by the transfectedcells disclosed above, and purified using standard techniques. (See forexample, Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press.)) The protein can thus be purifiedfrom prokaryotic or eukaryotic sources. In various further preferredembodiments, the protein is purified from bacterial, yeast, or mammaliancells. In a preferred embodiment, substantially purified means that thepolypeptide is substantially free of gel agents, such as polyacrylamideand agarose. In a further preferred embodiment, “substantially purified”means that they are free of other GPBP isoforms. In a further preferredembodiment, the substantially purified proteins are present in solution.As used herein, the term “substantially free of other proteins” meansthat contaminating proteins make up no more than about 5% of thesubstantially purified sample, preferably no more than about 3%.

In another embodiment of this aspect of the invention, the substantiallypurified polypeptide comprises or consists of an amino acid sequenceaccording to the genus R1-R2-R3, wherein

R1 is 0-90 amino acids of SEQ ID NO:35;

R2 is the amino acid sequence according to SEQ ID NO:29; and

R3 is an amino acid sequence selected from the group consisting of SEQID NO: 2 and SEQ ID NO:4.

In this embodiment, the R1 position is variable, and can be 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 amino acids ofSEQ ID NO:35. If R1 is 90 amino acid residues of SEQ ID NO:35 theresulting polypeptide comprises the polypeptide of SEQ ID NO:6 or SEQ IDNO:8, depending on the identity of the R3 group. Based on the aboveteachings, the various polypeptides encompassed by this R1 embodimentwill be apparent to one of skill in the art.

In another embodiment, the substantially purified polypeptide comprisesor consists of a polypeptide of the genus X1-X2, wherein:

X1 is 0-90 amino acids of SEQ ID NO:35;

X2 is the amino acid sequence according to SEQ ID NO:29 wherein thepolypeptide does not include the sequence of SEQ ID NO:2 or SEQ ID NO:4.

In this embodiment, the R1 position is variable, and can be 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90 amino acids ofSEQ ID NO:35. If R1 is 90 amino acid residues of SEQ ID NO:35, theresulting polypeptide comprises the polypeptide of SEQ ID NO:34. Basedon the above teachings, the various polypeptides encompassed by this R1embodiment will be apparent to one of skill in the art.

In this embodiment, the substantially purified polypeptides providetools to distinguish between the different isoforms of GPBP identifiedherein. For example, the substantially purified polypeptides accordingto this embodiment can be used to generate antibodies that selectivelybind to the 91 kDa GPBP and that do not bind to the 77 kDa GPBP. Suchantibodies will be of utility, for example, in immunodetection assays asdescribed below.

The substantially purified polypeptides of the invention can be made byany method known to those of skill in the art, but are preferably madeby recombinant means based on the teachings provided herein. Forexample, a coding region of interest as disclosed herein can be clonedinto a recombinant expression vector, which can then be used totransfect a host cell for recombinant protein production by the hostcells.

“Recombinant expression vector” includes vectors that operatively link anucleic acid coding region or gene to any promoter capable of effectingexpression of the gene product. The promoter sequence used to driveexpression of the disclosed nucleic acid sequences in a mammalian systemmay be constitutive (driven by any of a variety of promoters, includingbut not limited to, CMV, SV40, RSV, actin, EF) or inducible (driven byany of a number of inducible promoters including, but not limited to,tetracycline, ecdysone, steroid-responsive). The construction ofexpression vectors for use in transfecting prokaryotic cells is alsowell known in the art, and thus can be accomplished via standardtechniques. (See, for example, Sambrook, Fritsch, and Maniatis, in:Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998Catalog (Ambion, Austin, Tex.)

The expression vector must be replicable in the host organisms either asan episome or by integration into host chromosomal DNA. In a preferredembodiment, the expression vector comprises a plasmid. However, theinvention is intended to include other expression vectors that serveequivalent functions, such as viral vectors.

The protein may comprise additional sequences useful for promotingpurification of the protein, such as epitope tags and transport signals.Examples of such epitope tags include, but are not limited to FLAG(Sigma Chemical, St. Louis, Mo.), myc (9E10) (Invitrogen, Carlsbad,Calif.), 6-His (Invitrogen; Novagen, Madison, Wis.), and HA (BoehringerManheim Biochemicals). Examples of such transport signals include, butare not limited to, export signals, secretory signals, nuclearlocalization signals, and plasma membrane localization signals.

As disclosed below, the inventors have further discovered that at leastthe 91-kDa GPBP enters into the cell secretory pathway, reaches theendosomal/lysosomal compartment and undergoes proteolysis to yieldproducts of lower molecular mass. Thus, in another embodiment, thepolypeptides of the present invention are substantially purifiedprocessed GPBP polypeptides derived from a precursor polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and/or SEQ ID NO:8 wherein thesubstantially purified polypeptide is reactive with an antibodyselective for one or more epitopes within one or more of the GPBPisoforms disclosed herein, wherein the substantially purified processedGPBP polypeptide is selected from the group consisting of:

(a) a 60 kDa GPBP with a molecular weight of approximately 60 kDa indenaturing gel electrophoresis, wherein the 60 kDa GPBP is present inlysosomes, cytoplasm, microsomes, and mitochondria in liver tissue,wherein the 60 kDa GPBP is membrane-associated or soluble in thelysosomes in liver tissue;

(b) a 44-47 kDa GPBP with a molecular weight of approximately 44-47 kDain denaturing gel electrophoresis, wherein the 44-47 kDa GPBP is presentin lysosomes in liver tissue, wherein the 44-47 kDa GPBP ispredominately formed through a leupeptin-sensitive proteolysis in livertissue;

(c) a 32 kDa GPBP with a molecular weight of approximately 32 kDa indenaturing gel electrophoresis, wherein the 32 kDa GPBP is present incytoplasm, mitochondria, microsomes, and lysosomes in liver tissue, andwherein the 32 kDa GPBP is formed through a leupeptin-insensitiveproteolysis in liver lysosomes.

As used herein, being of an approximate molecular weight as determinedby denaturing gel electrophoresis means that the polypeptide is within0-10% of the recited molecular weight, more preferably within 0-5%, andeven more preferably within 0-3% under the following gel conditions

As used herein, determination of molecular weights is as would bedetermined under the following conditions: sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) performed on MiniProteanIII (Bio-Rad) using polyacrylamide gels (29.2:0.8acrylamide:bisacrylamide) at room temperature and constant voltage (200volts); running buffer of 192 mM glycine, 24.7 mM Tris and 1% SDS;stacking gel of 3.65% acrylamide/bisacrylamide 122 mM HCl-Tris pH 6.8,0.1% SDS, 0.146% ammonium persulfate, 0.146% Temed; running gel of 10%acrylamide/bisacrylamide, 373 mM HCl-Tris pH 8.8; 0.1% SDS, 0.1%ammonium persulfate, 0.1% Temed; and samples were 31.25 mM HCl-Tris pH6.8, 5.16% glycerol, 1% SDS and 2.5% β-mercaptoethanol.

This range represents a standard fluctuation for such molecular weightdeterminations based on differences in gel reagents, running time,temperature, and voltage, and other variables as would be recognized bythose of skill in the art.

As used herein, the recitation of a processed GPBP being in a specificsubcellular compartment in liver tissue means that the protein ispresent in detectable levels in the recited cellular compartment, anddoes not mean that it is not present in detectable levels in othercellular compartments.

As used herein, being “membrane-associated” means that, in extracts ofthe subcellular extract being analyzed, detectable levels of thepolypeptide of interest are found in the membrane fraction insubcellular fractions isolated according to the methods disclosed below.

As used herein, “leupeptin-sensitive” means that, in the presence ofsufficient quantities of leupeptin, production of the recitedproteolytic product is reduced. As used herein, “leupeptin-insensitive”means that, in the presence of similar quantities of leupeptin as above,production of the recited proteolytic product is not reduced. Preferredembodiments for determining leupeptin-sensitivity are as described belowin the experimental section.

The substantially purified processed GPBP polypeptides of thisembodiment can be produced, for example, by a method comprising (a)providing cells that express one or more polypeptide comprising orconsisting of an amino acid sequence selected from the group consistingof SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8; (b) lysingthe cells and isolating one or more fractions of the cells comprisingfractions selected from the group consisting of cytoplasmic-containingfractions, mitochondrial-containing fractions, microsomal-containingfractions, and lysosomal-containing fractions; (c) contacting theisolated fractions with an immunoaffinity column comprising an antibodythat selectively binds to a polypeptide comprising or consisting of anamino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 underconditions that result in binding of one or more of the 60 kDa GPBP, the44-47 kDa GPBP, and the 32 kDa GPBP to the immunoaffinity column; (d)washing the column under conditions that remove cellular contents thatdo not selectively bind to the immunoaffinity column; (e) eluting thebound material from the immunoaffinity column to provide an eluate; and(f) size fractionating the eluate and isolating one or more of thefractions consisting of the approximately 60 kDa fraction, theapproximately 44-47 kDa fraction, and the approximately 32 kDa fraction,wherein the approximately 60 kDa fraction contains the substantiallypurified 60 kDa GPBP; the approximately 44-47 kDa fraction contains thesubstantially purified 44-47 kDa GPBP, and the approximately 32 kDafraction contains the substantially purified 32 kDa GPBP.

In a preferred embodiment of this method, the cells express at least onepolypeptide comprising or consisting of an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 6 and SEQ ID NO:8, morepreferably SEQ ID NO:6.

Antibodies for use in these methods include those described herein aswell as in WO 00/50607 and WO 02/061430. Cell fractionation,immunoaffinity column chromatography, size fractionation, and suitablewash and elution conditions are known to those of skill in the art.

In another embodiment, the substantially purified processed GPBPpolypeptides of this embodiment can be produced by a method comprising(a) providing cells that express one or more recombinant polypeptidescomprising or consisting of an amino acid sequence selected from thegroup consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ IDNO:8; (b) lysing the cells and obtaining a partially purified cellextract containing the recombinant polypeptides; (c) contacting thepartially purified cell extract with liver lysosomal extracts underconditions that promote processing of the recombinant polypeptides toproduce a processed extract; (d) contacting the processed extract withan immunoaffinity column comprising an antibody that selectively bindsto an epitope within the recombinant polypeptides and/or their processedforms comprising or consisting of an amino acid sequence selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35 under conditions that result inbinding of recombinant polypeptides and their processed forms to theimmunoaffinity column; (e) washing the column under conditions thatremove cellular contents that do not selectively bind to theimmunoaffinity column; (f) eluting the bound material from theimmunoaffinity column to provide an eluate; and (g) size fractionatingthe eluate and isolating one or more of the fractions consisting of theapproximately 60 kDa fraction, the approximately 44-47 kDa fraction, andthe approximately 32 kDa fraction, wherein the approximately 60 kDafraction contains the substantially purified 60 kDa GPBP; theapproximately 44-47 kDa fraction contains the substantially purified44-47 kDa GPBP, and the approximately 32 kDa fraction contains thesubstantially purified 32 kDa GPBP.

In a preferred embodiment of this method, the cells express at least onepolypeptide comprising or consisting of an amino acid sequence selectedfrom the group consisting of SEQ ID NO:6 and SEQ ID NO:8, morepreferably SEQ ID NO:6.

For this embodiment, one of skill in the art can use the teachings ofthe invention to prepare recombinant expression vectors expressing therecited polypeptides. Preparing cell extracts and liver lysosomalextracts are known to those in the art, and are further described below.

In a further embodiment, the polypeptides of the present inventioninclude an isolated polypeptide consisting of the amino acid sequence ofSEQ ID NO:38 (AA 1-299 of SEQ ID NO:2). This polypeptide is a truncatedversion of the 77 kDa GPBP. As described below, this polypeptide isdemonstrated to have a greater kinase activity under acidic conditionsthan GPBP, and thus may be functionally similar to the GPBP formspresent in the lysosome.

In a further aspect, the present invention provides pharmaceuticalcompositions comprising one or more substantially purified polypeptideas described above and a pharmaceutically acceptable carrier. In anon-limiting example, the pharmaceutical compositions of this aspect ofthe invention can be used for immunization to prepare antibodiesspecific for non-canonical GPBP isoforms, which themselves can be usedas therapeutics to modulate GPBP activity. Alternatively, thepharmaceutical compositions according to this aspect of the inventioncan themselves be used as therapeutics to inhibit GPBP activity in asubject in need thereof.

In another aspect, the present invention provides antibodies thatselectively bind to the substantially purified polypeptides disclosedherein, but which do not selectively bind to the peptide sequencePRSARCQARRRRGGRTSS (SEQ ID NO:103).

In a preferred embodiment, the antibodies of the invention selectivelybind to an epitope present within the GPBP isoforms disclosed herein anddo not selectively bind to a polypeptide consisting of the amino acidsequence of SEQ ID NO:2 or SEQ ID NO:4. In this embodiment, it isfurther preferred that the antibodies selectively bind to one or moreproteins comprising or consisting of a sequence selected from the groupconsisting of SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ IDNO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and/or toan epitope within one or more polypeptides selected from the groupconsisting of the 60 kDa GPBP, the 44-47 kDa GPBP, and the 32 kDa GPBP.Such antibodies can be produced by immunization of a host animal witheither the complete GPBP isoforms disclosed herein or with antigenicpeptides thereof, while selecting against those that selectively bind toSEQ ID NO:103, and/or to SEQ ID NO:2 and/or SEQ ID NO:4 (via, forexample, adsorption of such antibodies on an affinity column comprisingthe polypeptide of SEQ ID NO:103, SEQ ID NO:2 and/or SEQ ID NO:4). In apreferred embodiment, the antibodies selectively bind to an epitopewithin an amino acid sequence selected from the group consisting of SEQID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35. These sequences are not included in the sequence ofSEQ ID NO:2 or SEQ ID NO:4, and thus antibodies directed againstepitopes within these sequences do not selectively bind to SEQ ID NO:2or SEQ ID NO:4. Suitable antibodies include polyclonal, monoclonal, andhumanized monoclonal antibodies.

In a further embodiment, the antibodies selectively bind to an isolatedpolypeptide consisting of the amino acid sequence of SEQ ID NO:38 (AA1-299 of SEQ ID NO:2 or SEQ ID NO:4). This polypeptide is a truncatedversion of the 77 kDa GPBP or GPBPΔ26. As described below, thispolypeptide is demonstrated to have a greater kinase activity underacidic conditions than GPBP, and thus may be functionally similar to theGPBP forms present in the lysosome.

As used herein, the term “selectively bind(s)” means that the antibodiespreferentially bind to the polypeptide in question in a mixture ofpolypeptides.

In a further aspect, the present invention provides methods for makingantibodies selective for one or more GPBP isoforms, comprisingimmunizing a host animal with an antigenic epitope derived from apolypeptide consisting of an amino sequence selected from the groupconsisting of SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32,SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:101 andisolating antibodies from the host animal that selectively bind to thepolypeptide, wherein the isolated antibodies are selective for one ormore Goodpasture antigen binding protein isoforms.

Antibodies can be made by well-known methods, such as described inHarlow and Lane, Antibodies; A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., (1988). In one example, preimmuneserum is collected prior to the first immunization. A substantiallypurified polypeptide of the invention, or antigenic fragments thereof,together with an appropriate adjuvant, are injected into an animal in anamount and at intervals sufficient to elicit an immune response. Animalsare bled at regular intervals, preferably weekly, to determine antibodytiter. The animals may or may not receive booster injections followingthe initial immunization. At about 7 days after each boosterimmunization, or about weekly after a single immunization, the animalsare bled, the serum collected, and aliquots are stored at about −20° C.Polyclonal antibodies against the proteins and peptides of the inventioncan then be purified directly by passing serum collected from the animalthrough a column to which non-antigen-related proteins prepared from thesame expression system without GPBP-related proteins bound.

Monoclonal antibodies can be produced by obtaining spleen cells from theanimal. (See Kohler and Milstein, Nature 256, 495-497 (1975)). In oneexample, monoclonal antibodies (mAb) of interest are prepared byimmunizing inbred mice with the proteins or peptides of the invention,or an antigenic fragment thereof. The mice are immunized by the IP or SCroute in an amount and at intervals sufficient to elicit an immuneresponse. The mice receive an initial immunization on day 0 and arerested for about 3 to about 30 weeks. Immunized mice are given one ormore booster immunizations of by the intravenous (IV) route.Lymphocytes, from antibody positive mice are obtained by removingspleens from immunized mice by standard procedures known in the art.Hybridoma cells are produced by mixing the splenic lymphocytes with anappropriate fusion partner under conditions which will allow theformation of stable hybridomas. The antibody producing cells and fusionpartner cells are fused in polyethylene glycol at concentrations fromabout 30% to about 50%. Fused hybridoma cells are selected by growth inhypoxanthine, thymidine and aminopterin supplemented Dulbecco's ModifiedEagles Medium (DMEM) by procedures known in the art. Supernatant fluidsare collected from growth positive wells and are screened for antibodyproduction by an immunoassay such as solid phase immunoradioassay.Hybridoma cells from antibody positive wells are cloned by a techniquesuch as the soft agar technique of MacPherson, Soft Agar Techniques, inTissue Culture Methods and Applications, Kruse and Paterson, Eds.,Academic Press, 1973.

“Humanized antibody” refers to antibodies derived from a non-humanantibody, such as a mouse monoclonal antibody. Alternatively, humanizedantibodies can be derived from chimeric antibodies that retains orsubstantially retains the antigen-binding properties of the parental,non-human, antibody but which exhibits diminished immunogenicity ascompared to the parental antibody when administered to humans. Forexample, chimeric antibodies can comprise human and murine antibodyfragments, generally human constant and mouse variable regions. Sincehumanized antibodies are far less immunogenic in humans than thenon-human monoclonal antibodies, they are preferred for therapeuticantibody use.

Humanized antibodies can be prepared using a variety of methods known inthe art, including but not limited to (1) grafting complementaritydetermining regions from a non-human monoclonal antibody onto a humanframework and constant region (“humanizing”), and (2) transplanting thenon-human monoclonal antibody variable domains, but “cloaking” them witha human-like surface by replacement of surface residues (“veneering”).These methods are disclosed, for example, in, e.g., Jones et al., Nature321:522-525 (1986); Morrison et al., Proc. Natl. Acad. Sci., U.S.A.,81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988);Verhoeyer et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun.28:489-498 (1991); Padlan, Molec. Immunol. 31(3):169-217 (1994); andKettleborough, C. A. et al., Protein Eng. 4(7):773-83 (1991).

To generate an antibody response, the polypeptides of the presentinvention are typically formulated with a pharmaceutically acceptablecarrier for parenteral administration. Such acceptable adjuvantsinclude, but are not limited to, Freund's complete, Freund's incomplete,alum-precipitate, water in oil emulsion containing Corynebacteriumparvum and tRNA. The formulation of such compositions, including theconcentration of the polypeptide and the selection of the vehicle andother components, is within the skill of the art.

The term antibody as used herein is intended to include antibodyfragments thereof which are selectively reactive with the polypeptidesof the invention, or fragments thereof. Antibodies can be fragmentedusing conventional techniques, and the fragments screened for utility inthe same manner as described above for whole antibodies. For example,F(ab′)₂ fragments can be generated by treating antibody with pepsin. Theresulting F(ab′)₂ fragment can be treated to reduce disulfide bridges toproduce Fab′ fragments.

In a further aspect, the invention provides methods for detecting thepresence of one or more of the polypeptides of the invention in aprotein sample, comprising providing a protein sample to be screened,contacting the protein sample to be screened with an antibody againstone or more of the polypeptides of the invention, and detecting theformation of antibody-antigen complexes. In a preferred embodiment,methods for detecting the presence of a protein that is substantiallysimilar to one or more polypeptides comprising or consisting of an aminoacid sequence selected from the group consisting of SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, and/or a protein that issubstantially similar to one or more polypeptides selected from thegroup consisting of 60 kDa GPBP, 44-47 kDa GPBP, and 32 kDa GPBPcomprise

a) providing a protein sample to be screened;

b) contacting the protein sample to be screened with an antibodyselective for one or more of the GPBP isoforms disclosed herein underconditions that promote antibody-antigen complex formation; and

c) detecting the formation of antibody-antigen complexes, wherein thepresence of the antibody-antigen complex indicates the presence of aprotein comprising or consisting of a sequence that is substantiallysimilar to a sequence selected from the group consisting of SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ IDNO:33, SEQ ID NO:34, SEQ ID NO:35, and/or a protein that issubstantially similar to one or more polypeptides selected from thegroup consisting of 60 kDa GPBP, 44-47 kDa GPBP, and 32 kDa GPBP.

As used herein, the term “substantially similar” means that thepolypeptides share at least 70% amino acid identity along theirco-linear portions, and more preferably 75%, 80%, 85%, 90%, or 95%identity.

The antibody can be polyclonal, monoclonal, or humanized monoclonal asdescribed above, although monoclonal antibodies are preferred. As usedherein, the term “protein sample” refers to any sample that may containthe polypeptides of the invention, and fragments thereof, including butnot limited to tissues and portions thereof, tissue sections, intactcells, cell extracts, purified or partially purified protein samples,bodily fluids, and nucleic acid expression libraries. Accordingly, thisaspect of the present invention may be used to test for the presence ofthe non-canonical GPBP isoforms disclosed herein in these variousprotein samples by standard techniques including, but not limited to,immunolocalization, immunofluorescence analysis, Western blot analysis,ELISAs, and nucleic acid expression library screening, (See for example,Sambrook et al, 1989.) In one embodiment, the techniques may determineonly the presence or absence of the protein or peptide of interest.Alternatively, the techniques may be quantitative, and provideinformation about the relative amount of the protein or peptide ofinterest in the sample. For quantitative purposes, ELISAs are preferred.

Detection of immunocomplex formation between the polypeptides of theinvention, and their antibodies or fragments thereof, can beaccomplished by standard detection techniques. For example, detection ofimmunocomplexes can be accomplished by using labeled antibodies orsecondary antibodies. Such methods, including the choice of label areknown to those ordinarily skilled in the art. (Harlow and Lane, Supra).Alternatively, the antibodies can be coupled to a detectable substance.The term “coupled” is used to mean that the detectable substance isphysically linked to the antibody. Suitable detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials and radioactive materials. Examples of suitableenzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase. Examples of suitableprosthetic-group complexes include streptavidin/biotin andavidin/biotin. Examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Anexample of a luminescent material includes luminol. Examples of suitableradioactive material include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Such methods of detection are useful for a variety of purposes,including but not limited to detecting an autoimmune condition,identifying cells targeted for or undergoing apoptosis,immunolocalization of the proteins of interest in a tissue sample,Western blot analysis, and screening of expression libraries to findrelated proteins.

In another aspect, the present invention provides isolated nucleic acidsthat encode the truncated GPBP polypeptides of the invention. In oneembodiment, the isolated nucleic acids consist of sequences selectedfrom the group consisting of SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, and SEQ ID NO:27.

The isolated nucleic acid sequence may comprise RNA or DNA. As usedherein, “isolated nucleic acids” are those that have been removed fromtheir normal surrounding nucleic acid sequences in the genome or in cDNAsequences. Such isolated nucleic acid sequences may comprise additionalsequences useful for promoting expression and/or purification of theencoded protein, including but not limited to polyA sequences, modifiedKozak sequences, and sequences encoding epitope tags, export signals,and secretory signals, nuclear localization signals, and plasma membranelocalization signals.

In another aspect, the present invention provides recombinant expressionvectors comprising isolated nucleic acids consisting of a sequenceselected from the group consisting of SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, and SEQ ID NO:27. Recombinant expression vectorsare vectors that operatively link a nucleic acid coding region or geneto any promoter capable of effecting expression of the gene product thatare operably linked to a promoter, and are discussed in more detailabove.

In a further aspect, the present invention provides host cells that havebeen transfected with the recombinant expression vectors disclosedherein, wherein the host cells can be either prokaryotic or eukaryotic.The cells can be transiently or stably transfected. Such transfection ofexpression vectors into prokaryotic and eukaryotic cells can beaccomplished via any technique known in the art, including but notlimited to standard bacterial transformations, calcium phosphateco-precipitation, electroporation, or liposome mediated-, DEAE dextranmediated-, polycationic mediated-, or viral mediated transfection. (See,for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al.,1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: AManual of Basic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc.New York, N.Y.).

Methods for Identifying and Making Candidate Compounds to TreatAutoimmune Conditions and/or Protein Deposit-Mediated Disorders

GPBP displays a number of biological features to be considered a goodcandidate as a pivotal component of the cellular machinery catalyzingconformational isomerization and supramolecular assembly of autoantigensand inducing immune response during autoimmune pathogenesis (See below,as well as WO 00/50607; WO 02/061430). The results disclosed hereinsuggest that GPBP is an integral component of the endosomal-lysosomalpathway which activity is regulated in part by a catepsin-dependentprocessing, a biological strategy described for other enzymes (Pham, C.T., & T. J., Ley, (1999). Proc Natl Acad Sci USA 96(15): 8627-8632).These proteases are critical in processing proteins entering endosomalpathway and producing peptides that are presented through MHC class II(Chapman, H. A., (1998) Curr Opin Immunol 10(1): 93-102). Disturbance oflysosomal environment in a more general manner such as modifying the pHusing compounds as chloroquine or in a more specific manner usingcatepsin inhibitors such as leupeptin has been shown to alter peptidepresentation by MHC class II (Demotz, S., P. M. Matricardi, C. Irle, P.Panina, A. Lanzavecchia, & G. Corradin, (1989) J Immunol 143(12):3881-3886; Turk, V., B. Turk, & D. Turk, (2001) EMBO J 20(17):4629-4633). We have shown herein that leupeptin treatment substantiallyalters lysosomal processing of GPBP and therefore also likely induces analteration in GPBP activity, which in turn suggests that altered peptidepresentation and altered GPBP activity may be related and perhapscritical in autoimmune pathogenesis, which necessarily requires aberrantpeptide presentation to be effective.

A feature common to many degenerative diseases is the formation ofdeposits of specific polypeptides. Where and how these deposits appearis highly specific and tightly related with pathogenesis. The depositscan be nuclear inclusion bodies, as in cerebellar ataxia, or be at theER lumen, such as in some degenerative disease affecting liver andneurons, or be cytoplasmic inclusion bodies, as in Parkinson's disease,Alzheimer's disease, and amyotrophic lateral sclerosis; orendosomal-lysosomal, as in Alzheimer's disease, prion diseases, and typeII diabetes. GPBP is an ubiquitous protein that has been independentlyrelated to conformational catalysis of substrate proteins (WO 00/50607;WO 02/061430) and in the formation of protein deposits in animal modelsthat develop a degenerative nephropaty associated to an autoimmuneresponse. Consequently the finding disclosed herein that GPBP interactswith PrP and Aβ₁₋₄₂ two polypeptides that undergo conformationalalteration and form amyloid deposits in prion diseases and Alzheimer'sdisease, respectively, represents strong evidence for GPBP beinginvolved in the pathogenesis of these degenerative diseases. Morespecifically, a protein resident in the endosomal-lysosomal pathwaynamed Protein X has been proposed to bind to PrP and catalyze theconformational transition from PrP^(C) to Prp^(Sc) (Prusiner, S. B.,(1998). “Prions.” Proc Natl Acad Sci USA 95(23): 13363-13383.). Hereinwe present evidence demonstrating that GPBP binds to PrP in a Protein Xfashion, phosphorylates PrP, forms aggregates with it and, as aconsequence of this interaction, PrP undergoes conformational changesthat renders PrP highly insoluble and precipitable. To our knowledge,GPBP represents the best molecular candidate to be Protein X in priondiseases as well as to perform a similar role in other proteindeposit-mediated human disease.

Thus, in another aspect, the present invention provides methods foridentifying compounds to treat an autoimmune disorder, wherein themethod comprises identifying compounds that inhibit activity of one ormore GPBP isoforms of the present invention. In a preferred embodiment,the one or more GPBP isoform comprises or consists of a sequenceselected from the group consisting of SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, or one ormore GPBP isoforms selected from the group consisting of 60 kDa GPBP,44-47 kDa GPBP, and 32 kDa GPBP, wherein such compounds are candidatecompounds for treating an autoimmune condition and/or proteindeposit-mediated disorders.

In another aspect, the present invention provides methods foridentifying compounds to treat a protein deposit-mediated condition,wherein the method comprises identifying compounds that inhibit activityof one or more GPBP isoforms of the present invention. In a preferredembodiment, the one or more GPBP isoforms comprises or consists of asequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4,SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, or are selected from the group consisting of 60 kDaGPBP, 44-47 kDa GPBP, and 32 kDa GPBP, wherein such compounds arecandidate compounds for treating an autoimmune condition and/or proteindeposit-mediated disorders.

In a further embodiment of these methods, the method further comprisesmaking the compounds identified as candidate compounds for treating anautoimmune condition and/or protein deposit-mediated disorders. In oneexample, such compounds are organic molecules that are made usingstandard chemical methods. In another example, such compounds arepolypeptides, which are made by methods disclosed herein for makingpolypeptides.

In one embodiment of these aspects, the method comprises identifyingcompounds that inhibit GPBP kinase activity. Such inhibition can beinhibition of GPBP autophosphorylation and/or inhibition of GPBPphosphorylation of target polypeptide, such as α3(IV) NC1 domain, myelinbasic protein, and prion protein. Examples of such target polypeptidescomprise those provided as SEQ ID NO:52 (α3(IV)NC1); SEQ ID NO:53 (MBP);and SEQ ID NO:54 (PrP), or functional equivalents. In a furtherembodiment, the method comprises identifying compounds that inhibit GPBPcatalysis of conformational isomerization of a target polypeptide, suchas α3(IV) NC1 domain, myelin basic protein, prion protein, and Aβ₁₋₄₂.Examples of such polypeptides are as described above, also polypeptidescomprising SEQ ID NO:55 (Aβ₁₋₄₂), and functional equivalents thereof.Those of skill in the art will be able to identify other targetpolypeptides that can be used in the methods of the invention, such asSEQ ID NO:102, a functional equivalent for MBP.

In a further embodiment of these aspects, the method comprisesidentifying compounds that inhibit both GPBP kinase activity and GPBPcatalysis of conformational isomerization of a target polypeptide.

The phosphorylation assays can be conducted in vitro on isolatedtargets, or can comprise analyzing the effects of the one or more testcompounds on phosphorylation in cultured cells, although in vitro assaysare preferred. A preferred method for identifying compounds that reducein vitro phosphorylation of the target polypeptide comprises incubatinga target polypeptide and ATP in vitro in the presence or absence of oneor more test compounds under conditions that promote phosphorylation ofthe target polypeptide in the absence of the one or more test compounds;detecting phosphorylation of the target polypeptide; and identifyingtest compounds that reduce phosphorylation of the target polypeptiderelative to phosphorylation of the target polypeptide in the absence ofthe one or more test compounds.

One of skill in the art is capable of determining suitablephosphorylation conditions for conducting the phosphorylation assay, andthus the present method is not limited by the details of the particularphosphorylation conditions employed. A non-limiting example of suchsuitable conditions for assaying phosphorylation of the first targetcomprises the use of 25 mM β-glycerol phosphate pH 7, 0.5 mM EGTA, 8 mMMg Cl₂, 5 mM MnCl₂, 1 mM DTT, y 0.132 μM [γ³²P]-ATP using 100-200 μg ofenzyme and 1 μg of substrate at variable time at 30° C.

In one embodiment of these aspects, the target polypeptide is GPBP, andthe assay comprises analyzing the effect(s) of the one or more testcompounds on GPBP autophosphorylation. In such an embodiment, anexemplary amount of GPBP for use in the assay is between 50 to 200 ng.In an alternative embodiment, the target polypeptide is selected fromthe group consisting of an α3 type IV collagen NC1 domain polypeptidecomprising the amino acid sequence of SEQ ID NO:52, an MBP polypeptidecomprising the amino acid sequence of SEQ ID NO:53, and a prion protein,such as that in SEQ ID NO:54 and the assay is conducted in the presenceof a GPBP isoform as recited above, to test for transphosphorylation ofthe target polypeptide by the protein kinase. In this embodiment, thetarget polypeptide can comprise a full length α3 type IV collagen NC1domain polypeptide (including α3(IV)NC1Asp⁹ SEQ ID NO:57 orα3(IV)NC1Ala⁹ SEQ ID NO:56), full length MBP, and prion protein, orportions thereof that contain sequences sufficient for phosphorylationby GPBP.

For in vitro phosphorylation assays, detection of phosphorylation can beaccomplished by any number of means, including but not limited to using³²P labeled ATP and carrying out autoradiography of a Western blot ofthe resulting protein products on a reducing or non-reducing gel, or byscintillation counting after a step to separate incorporated fromunincorporated label.

Analysis of in vitro phosphorylation may further include identifying theeffect of the one or more test compounds on phosphorylation ofindividual conformational isomers of the target polypeptide. Suchidentification can be accomplished, for example, by carrying outSDS-PAGE on the reaction products of the phosphorylation reaction,followed by Western blotting, autoradiography and immunodetection of thetarget protein, as disclosed in WO 02/061430.

Analysis of in vitro phosphorylation may further include identifying theeffect of the one or more test compounds on Ser⁹ phosphorylation of theα3 type IV collagen NC1 domain, as disclosed in WO 02/061430. Suchidentification can be accomplished, for example, by comparing theimmunoreactive patterns of antibodies specifically reacting with the Nterminus of the α3(IV)NC1 (including but not limited to antibodiesdisclosed in WO 02/061430) and antibodies specifically reacting withSer(P), such as those commercially available from Sigma Chemical Co.(St. Louis, Mo.).

The data presented in WO 02/061430 suggest that phosphorylation at Ser⁹exerts a positive control over conformational isomerization of α3(V)NC1,and efficiently changes the cohort of α3(IV)NC1 conformers produced by acell. These findings suggest that Ser⁹ is one of the structural featuresthat renders the α3(IV)NC1 domain potentially immunogenic, and suggestthat, during pathogenesis, an aberrant phosphorylation event on thisserine can lead the formation of conformers for which the immune systemhas not established a tolerance. Thus, determining the effect of testcompounds on phosphorylation of the Ser⁹ residue of α3 type IV collagenNC1 domain may be important in identifying especially useful candidatecompounds for treating autoimmune disorders. Ser⁸ in MBP has been shownto be functionally similar to Ser⁹ in α3(IV)NC1 conformation andtherefore similar tests can be conducted to identify compounds affectingMBP Ser⁸ phosphorylation. (See WO 00/50507 and WO 02/061430)

Alternatively, the effects of test compounds on phosphorylation of thetarget polypeptide can be analyzed in cultured cells. Such a methodinvolves contacting cells that express a target polypeptide selectedfrom the group consisting of an α3 type IV collagen NC1 domainpolypeptide, MBP, and prion protein under conditions to promotephosphorylation, detecting phosphorylation of the target polypeptide;and identifying test compounds that reduce phosphorylation of the targetpolypeptide relative to phosphorylation of the target polypeptide in theabsence of the one or more test compounds. Appropriate cells for use areeukaryotic cells that express the appropriate target protein. Methods ofdetecting phosphorylation are as described above.

As used herein, the phrase “reduce/reducing phosphorylation” means tolessen the phosphorylation of the target polypeptide relative tophosphorylation of the target polypeptide in the absence of the one ormore test compounds. Such “reducing” does not require elimination ofphosphorylation, and includes any detectable reduction inphosphorylation. Thus, a test compound that inhibits phosphorylation ofthe target by, for example, as little as 10-20% would be considered atest compound that reduced phosphorylation. Such a compound may, forexample, affect phosphorylation of Ser⁹ of the α3(IV) NC1 polypeptide orSer⁸ in MBP, which is shown to exert a powerful control onconformational diversification, and thus to be a strong candidate for aninhibitor of autoimmunity. Alternatively, a test compound may inhibitphosphorylation of target polypeptide by 90%, but have little inhibitoryeffect on conformational isomerization of the target polypeptide,because reduction affects phosphorylation at sites other than Ser⁹ orSer⁸. By performing assays both for phosphorylation inhibition of thetarget polypeptide, and conformer inhibition of the target polypeptide,it is possible to identify those compounds with the best potential foruse as therapeutics for autoimmune disorders.

The above methods can be performed in whole cells or cell extractsexpressing recombinant or naturally occurring forms of the polypeptides,in the absence of cells using proteins isolated via any of the methodsdisclosed herein and optionally including lysosomal extracts, or via anyother methods known in the art.

Similarly, inhibition of conformational isomerization of the targetpolypeptide can be carried out in vitro using isolated components, orcan be carried out in cultured cells, although the use of cultured cellsis preferred. In a preferred embodiment using cultured cells,identifying compounds that reduce formation of conformational isomers ofthe target polypeptide comprises:

(a) providing cells that express a target polypeptide selected from thegroup consisting of α3(IV)NC1 domain, MBP, prion protein, Aβ1-42 andfunctional equivalents thereof

(b) contacting the cells with one or more GPBP isoforms comprising orconsisting of an amino acid sequence selected from the group consistingof SEQ ID NO:2 (for identifying compounds for treating a proteindposit-mediated disorder), SEQ ID NO:4 (for identifying compounds fortreating a protein dposit-mediated disorder), SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, orone or more GPBP isoforms selected from the group consisting of 60 kDaGPBP, 44-47 kDa GPBP, and 32 kDa GPBP;

(c) contacting the cells in the presence or absence of one or more testcompounds, under conditions that promote conformational isomerization ofthe target polypeptide catalyzed by the one or more GPBP isoforms in theabsence of the one or more test compounds, wherein the contacting of thecells with the one or more test compounds can occur prior to,simultaneous with, or subsequent to contacting the cells with the one ormore GPBP isoforms;

(d) detecting conformational isomerization of the target polypeptide;and

iv) identifying test compounds that reduce conformational isomerizationof the target polypeptide relative to conformational isomerization ofthe target polypeptide in the absence of the one or more test compounds.

Appropriate cells for use are eukaryotic cells that express theappropriate target polypeptide. In a preferred embodiment, cell linesstably transfected to express the target polypeptide are used.

In this embodiment, detection of conformational isomers of the targetpolypeptide, and the effects of the test compounds thereon, generallyinvolve immunodetection using Western blots of non-reducing SDS-PAGEgels containing the polypeptides from the cells. The target polypeptidecan be purified via standard techniques (such as using cells transfectedwith a recombinant target polypeptide that is linked to an epitope tagor other tag to facilitate purification), or cell extracts can beanalyzed. In a most preferred embodiment, stable cell lines (such asthose disclosed in WO 02/061430) expressing recombinant targetpolypeptide are used. In some cases, such as for the α3 type IV collagenNC1 domain polypeptide, the target polypeptide is secreted into themedium in a monomeric form, permitting running of serum-free mediasamples on SDS-PAGE gels and subsequent Western blot analysis andimmunodetection. Alternatively, protein extracts from the cells can bemade by standard techniques. In a further alternatively, serum freemedia or otherwise isolated proteins can be used to coat ELISA plates,followed by similar immunodetection using antibodies that selectivelybind to native conformers and either aberrant conformers or allconformers, respectively, and analysis using plate readers.

In a further embodiment, a reduction in conformational isomerization isdetermined by first subjecting the samples (in vitro reactions orcultured cells) to centrifugation and using the supernatant for limitedproteolysis and further analysis of products by either Western blot ormass spectrometry. Alternatively, supernatants can be analyzed by ELISAusing monoclonal antibodies that recognize conformational epitopes ofthe target protein. In this embodiment, it is possible to distinguishbetween a reduction in conformational isomerization and reduction ofrandom aggregation, since the supernatant is used to analyzeconformational isomerization, while the precipitate is used to analyzerandom aggregation, as described below.

In a preferred embodiment of an in vitro assay for inhibitors ofconformational isomerization of the target polypeptide, the methodcomprises incubating

(a) a target polypeptide selected from the group consisting of α3(IV)NC1domain, MBP, and prion protein, and functional equivalents thereof

(b) a GPBP isoform comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO:2 (for identifying compounds for treatinga protein dposit-mediated disorder), SEQ ID NO:4 (for identifyingcompounds for treating a protein dposit-mediated disorder), SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28; or a GPBP isoform selected from the group consisting of 60 kDaGPBP, 44-47 kDa GPBP, and 32 kDa GPBP; in the presence or absence of oneor more test compounds, under conditions that promote conformationalisomerization of the target polypeptide catalyzed by the GPBP isoform inthe absence of the one or more test compounds, detecting conformationalisomerization of the target polypeptide; and identifying test compoundsthat reduce conformational isomerization of the target polypeptiderelative to conformational isomerization of the target polypeptide inthe absence of the one or more test compounds, wherein such compoundsare candidate compounds to treat one or more of an autoimmune conditionand a protein deposit-mediated disorder.

As used herein, the phrase “reduce/reducing conformationalisomerization” means to lessen the formation of conformers of the targetpolypeptide relative to conformer production under control conditions.Such “reducing” does not require elimination of conformer formation, andincludes any detectable reduction in conformer formation. Furthermore,such “reduction in conformer formation” may entail a reduction in onlyone, or fewer than all conformational isomers; one can envision thatsuch a reduction in production of specific conformers may be accompaniedby an increase in the formation of other conformers. For example, wepresent evidence in WO 02/061430 that, for the α3(IV) NC1 domainpolypeptide, a 27 kDa conformer is the primary product from which theremaining conformers derive. Thus, in a further preferred embodiment,the method comprises identifying those compounds that do not alter theformation of the 27-kDa conformer, but reduce formation of one or moreof the other conformers. A preferred method for monitoring thisinhibition of specific conformers is to use Mab3 antibody (described inWO 02/061430), which only reacts with the 27-kDa conformer, in parallelwith Mab175, which is equally reactive with all a3 type IV collagen NC1domain conformers.

In a further preferred embodiment of the assays to identify inhibitorsof conformational isomerization of the target polypeptide, the targetpolypeptide is an α3(IV)NC1 domain polypeptide, and analysis of testcompound effect on conformer formation of each of wild type α3(IV)NC1and α3(IV)NC1Asp⁹ (SEQ ID NO:57) is carried out in parallel.α3(IV)NC1Asp⁹ is modified to replace Ser⁹ with Asp⁹, an amino acidresidue that mimics a permanently phosphorylated residue, which is usedherein as an example of an aberrant phosphorylation of α3(IV)NC1, thatleads to the production of aberrant conformers. In WO 02/061430, we showthat α3(IV)NC1Asp⁹ expressing cells produce a larger number ofconformers than cells expressing α3(IV)NC1Ser⁹. Furthermoreα3(IV)NC1Asp⁹ cells express a 27-kDa conformer that reacts more stronglywith Mab3, as well as with Goodpasture patient autoantibodies, than the27-kDa conformer produced by α3(IV)NC1Ser⁹ expressing cells. It is mostpreferred to identify compounds that abolish these differences inconformer production between α3(IV)NC1Asp⁹ and α3(IV)NC1Ser⁹, becausethis will indicate that the compound inhibits the production of anaberrant 27-kDa conformer from α3(IV)NC1Asp⁹, while maintainingappropriate conformer production for α3(IV)NC1Ser⁹.

In a further preferred embodiment, identifying compounds for treating anautoimmune disorder further comprises identifying compounds that reducerandom aggregation of the target protein. As used herein, “randomaggregation” is defined as non-physiological protein aggregation, asopposed to non-random, physiological protein oligomerization. GPBPcatalyzes in vitro oligomerization and prevents random aggregation ofprotein substrates such as α3(IV)NC1.

While not being limited by a specific mechanism, we propose that theideal drug candidate for treating autoimmune disorders and/or proteindeposit-mediated disorders would inhibit the kinase and chaperonineactivity of GPBP, but would not inhibit its chaperone (ie: randomaggregate-disrupting) activity (See WO 02/061430), in order to minimizethe possibility that inhibition of GPBP activity would lead to increasedrandom aggregate formation. Even more preferably, the ideal drugcandidate would, in fact, enhance the chaperone activity of GPBP, tominimize secondary effects derived from undesirable aggregation ofconformers.

Both in vitro assays and assays utilizing cultured cells can be used foridentifying compounds that reduce random aggregation of the targetpolypeptide, although in vitro methods are preferred. One embodiment ofan in vitro assay comprises:

i) incubating in vitro a target polypeptide selected from the groupconsisting of α3(IV)NC1, MBP, prion protein, Aβ₁₋₄₂, and functionalequivalents thereof, with a GPBP isoform comprising an amino acidsequence selected from the group consisting of SEQ ID NO:2 (foridentifying compounds for treating a protein-deposit-mediated disorder),SEQ ID NO:4 (for identifying compounds for treating aprotein-deposit-mediated disorder), SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28; or a GPBPisoform selected from the group consisting of 60 kDa GPBP, 44-47 kDaGPBP, and 32 kDa GPBP in the presence or absence of one or more testcompounds, under conditions to promote random aggregation of the targetpolypeptide by the GPBP isoform in the absence of the one or more testcompounds; and

ii) identifying test compounds that reduce random aggregation of thetarget polypeptide by the GPBP isoform relative random aggregation ofthe target polypeptide by the GPBP isoform in the absence of the one ormore test compounds.

Detection of random aggregates, and the effect of test compoundsthereon, is preferably carried out by Western blotting of a non-reducingSDS-PAGE gel of the isolated target polypeptide after incubation, andprobing with antibodies that recognize the target polypeptide.Preferably, immunodetection is carried out using, in parallel, anantibody that detects a native conformation of the target polypeptide(such as Mab3 which selectively binds to an α3 type IV collagen NC1domain polypeptide conformer (WO 02/061430)), and an antibody thatdetects all target polypeptide conformational isomers (such as Mab175disclosed in WO 02/061430).

In a further embodiment, detection of random aggregation either in vitroor in cultured cells comprises centrifuging the samples and using theprecipitates for direct Western blot analysis or for specific limitedproteolysis followed by analysis of proteolytic products by eitherWestern blot analysis or mass spectrometry. In many cases this is apreferred embodiment, as random aggregates of the target protein aregenerally precipitable, and therefore centrifugation separates randomfrom non-random aggregates.

In a preferred embodiment of the random aggregation assay using culturedcells, cells that express the α3(IV)NC1 domain alone, the entire α3(IV)chain or type IV collagen containing α3(IV) chain are contacted with theone or more test compounds, and the α3(IV)NC1 domain or collagenasedigested α3(IV) chain or type IV collagen produced and secreted by thecells analyzed for α3(IV)NC1 oligomers by Western blot analysis asdescribed in WO 02/061430.

As used herein the phrase “reduce/reducing GPBP induced randomaggregation of the target polypeptide” means to decrease the amount ofGPBP induced random aggregates of the target polypeptide relative torandom aggregation under control conditions. Such “reducing” does notrequire elimination of random aggregation formation, and includes anydetectable reduction in random aggregation formation, includingreduction in only a single species of random aggregation in the presenceof increased in other species of random aggregates.

In a further embodiment, the method for identifying candidate compoundsto treat an autoimmune condition and/or a protein deposit-mediateddisorder comprises contacting: (a) a GPBP isoform comprising orconsisting of an amino acid sequence selected from the group consistingof SEQ ID NO:2 (for identifying compounds for treating proteindeposit-mediated disorders), SEQ ID NO:4 (for identifying compounds fortreating protein deposit-mediated disorders), SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28; ora GPBP isoform selected from the group consisting of 60 kDa GPBP, 44-47kDa GPBP, and 32 kDa GPBP with (b) a target polypeptide selected fromthe group consisting of α3(IV) NC1 domain, MBP, prion protein, Aβ1-42,and functional equivalents thereof, in the presence of one or more testcompounds, under conditions that promote formation of an interactionbetween the GPBP isoform and the target polypeptide in the absence oftest compounds and identifying test compounds that inhibit theinteraction, wherein such compounds are candidate compounds to treat anautoimmune condition and/or a protein deposit-mediated disorder.

Such methods can be performed in whole cells or cell extracts (such asmammalian brain extracts) expressing recombinant or naturally occurringforms of the GPBP isoform and/or the target polypeptide, in the absenceof cells using proteins isolated via any of the methods disclosed hereinand optionally including lysosomal extracts, or via any other methodsknown in the art. The interaction between the GPBP isoform and thetarget polypeptide can be monitored by a variety of methods, includingco-immunoprecipitation assays using antibodies directed against the GPBPisoform, the target polypeptide, and/or antibodies directed againstexpression tags added to recombinant versions of the GPBP isoform and/orthe target polypeptide. Alternatively interactions can be monitored byanalyzing aggregation kinetics as discussed below.

It should be noted that in each of the above embodiments of methods fordetecting candidate compounds for treating an autoimmune conditionand/or protein deposit-mediated disorders, conditions can be modified toreduce the pH of the reactions to approximate conditions in cellularcompartments to which various GPBP isoforms have been localized. Suchreaction conditions may better approximate physiological conditions. Forexample, a pH in the range of 5 to 5.5 could be used to simulateconditions in the lysosome or 6-6.5 to simulate conditions in theER/Golgi.

As used herein a “protein deposit-mediated disorder” means a diseasemediated by abnormal deposition of a specific protein, including but notlimited to Parkinson's disease, Alzheimer's disease, amyotrophic lateralsclerosis, prion diseases, and type II diabetes, and autoimmunedisorders. The protein deposit may be amyloid matter or para-amyloidmatter.

As used herein an “autoimmune condition” is selected from the groupconsisting of Goodpasture Syndrome, multiple sclerosis, systemic lupuserythematosus, cutaneous lupus erythematosus, pemphigus, pemphigoid andlichen planus.

Modulators of GPBP Activity

In another aspect, the present invention provides a method for treatingan autoimmune disorder, a tumor, a protein deposit-mediated disorder,and/or for preventing cell apoptosis comprising modification of theexpression or activity of a GPBP isoform comprising or consisting of anamino acid sequence selected from the group consisting of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:SEQ ID NO:26, SEQ ID NO:28; or a GPBP isoform selectedfrom the group consisting of 60 kDa GPBP, 44-47 kDa GPBP, and 32 kDaGPBP. Modifying the expression or activity of these polypeptides can beaccomplished by using inducers or inhibitors of GPBP expression oractivity, such as GPBP antibodies, antisense oligonucleotidescomplimentary to the transcription product of the GPBP gene, smallinterfering RNAs targeting the transcription product of the GPBP gene,gene or protein therapy using GP or myelin basic protein alternativeproducts, cell therapy using host cells expressing GP or myelin basicprotein alternative products, or other techniques known in the art. Asused herein, “modification of expression or activity” refers tomodifying expression or activity of either the RNA or protein product.Examples of such inducers or inhibitors are discussed below.

As part of the present invention, the inventors have identified furtherinhibitors of GPBP activity. Thus, in another aspect, the presentinvention provides an isolated polypeptide consisting of an amino acidsequence according to the general formula X1-SHCIX2-X3, wherein:

X1 is 0-10 amino acids of the sequence ATTAGILATL (SEQ ID NO:41);

X2 is E or Q; and

X2 is 0-10 amino acids of the sequence LMVKREDSWQ (SEQ ID NO:42).

As described below, the inventors have identified the peptide “SHCIE”(SEQ ID NO:39), which is derived from the GPBP sequence disclosed in WO00/50607, as a key site for self-interaction of GPBP. As such, use ofpeptides comprising this sequence has been shown to inhibit GPBP kinaseactivity which makes them useful as therapeutics for a number ofindications, as discussed below. A similar sequence (SHCIQ (SEQ IDNO:40)) is present in aggregatable CaM kinase II subunits α, β and δ,whereas it is not present in non-aggregatable CaM kinases I and IV (seebelow).

X1 and X3 provide optional amino acid sequences from GPBP immediatelyflanking the core sequence, to provide appropriate secondary structuralcharacteristics to the polypeptide for optimal inhibitory activity.

In a preferred embodiment of this aspect of the invention, thepolypeptide consists of a sequence selected from the group consisting ofSHCE (SEQ ID NO:39), SHCIQ (SEQ ID NO:40), ILATLSHCIELMVKR (SEQ IDNO:43), and ILATLSHCIQLMVKR (SEQ ID NO:44).

As described below, the inventors have further identified the peptideEKTAGKPILF (SEQ ID NO:45), present at the carboxy terminus of GPBP, asbeing a key site for GPBP self-interaction. As such, peptides of 6 ormore amino acids derived from this sequence are useful as therapeuticsfor a number of indications, as discussed below. Thus, in anotherembodiment, the present invention provides isolated polypeptidesconsisting of at least 6 amino acids of the sequence EKTAGKPILF (SEQ IDNO:45). In a preferred embodiment, the isolated polypeptide consists ofthe sequence EKTAGKPILF (SEQ ID NO:45).

The polypeptides according of this aspect of the invention can furtherbe derivatized to provide enhanced half-life, such as by the addition ofpolyethylene glycol (PEG) or as otherwise known in the art. Thepolypeptides of the invention may comprise L-amino acids, D-amino acids(which are resistant to L-amino acid-specific proteases in vivo), acombination of D- and L-amino acids, and various “designer” amino acids(e.g., β-methyl amino acids, Cα-methyl amino acids, and Nα-methyl aminoacids, etc.) to convey special properties. Synthetic amino acids includeornithine for lysine, and norleucine for leucine or isoleucine.

In addition, the polypeptides can have peptidomimetic bonds, such asester bonds, to prepare polypeptides with novel properties. For example,a polypeptide may be generated that incorporates a reduced peptide bond,i.e., R₁—CH₂—NH—R₂, where R₁ and R₂ are amino acid residues orsequences. A reduced peptide bond may be introduced as a dipeptidesubunit. Such a polypeptide would be resistant to protease activity, andwould possess an extended half-live in vivo.

The term “polypeptide” is used in its broadest sense to refer to asequence of subunit amino acids, amino acid analogs, or peptidomimetics.The subunits are linked by peptide bonds, although the polypeptide cancomprise further moieties that are not necessarily linked to thepolypeptide by a peptide bond. For example, as discussed above, thepolypeptide can further comprise a non-amino acid molecule that containsan aromatic ring.

The polypeptides described herein may be chemically synthesized orrecombinantly expressed. Recombinant expression can be accomplishedusing standard methods in the art, as disclosed above. Such expressionvectors can comprise bacterial or viral expression vectors, and suchhost cells can be prokaryotic or eukaryotic.

Preferably, the polypeptides for use in the methods of the presentinvention are chemically synthesized. Synthetic polypeptides, preparedusing the well-known techniques of solid phase, liquid phase, or peptidecondensation techniques, or any combination thereof, can include naturaland unnatural amino acids. Amino acids used for peptide synthesis may bestandard Boc (Nα-amino protected Nα-t-butyloxycarbonyl) amino acid resinwith standard deprotecting, neutralization, coupling and wash protocols,or standard base-labile Nα-amino protected 9-fluorenylmethoxycarbonyl(Fmoc) amino acids. Both Fmoc and Boc Nα-amino protected amino acids canbe obtained from Sigma, Cambridge Research Biochemical, or otherchemical companies familiar to those skilled in the art. In addition,the polypeptides can be synthesized with other Nα-protecting groups thatare familiar to those skilled in this art.

Solid phase peptide synthesis may be accomplished by techniques familiarto those in the art and provided, such as by using automatedsynthesizers.

In a further aspect, the present invention provides silencers of GPBPand/or GPBPΔ26 expression, selected from the group consisting ofsiGPBPΔ26-1 (SEQ ID NO:47), siGPBPΔ26-2 (SEQ ID NO:48), siGPBPΔ26-3 (SEQID NO:49), siGPBPΔ26-4 (SEQ ID NO:50), and siGPBP (SEQ ID NO:51). Thesenucleic acids may be DNA or RNA, and may be single stranded or doublestranded (in which case they also include the nucleic acid sequencecomplementary to the recited sequence, as well be recognized by those ofskill in the art), although they are preferably RNA and double stranded.When used as DNA they are delivered into the cell in an appropriatevector for intracellular transcription and double stranded RNAsynthesis, as is known in the art. As discussed below, each of thesesilencers was shown to diminish GPBP and/or GPBPΔ26 expression, and thusthey are useful for the therapeutic methods of the invention, asdiscussed below. The silencers can be made by standard methods, such asthose disclosed herein.

In a preferred embodiment, the nucleic acids are used in the methods forthe invention as double stranded RNAs. Methods for using such doublestranded RNAs are as described, for example in U.S. Pat. No. 6,506,559.For example, RNA may be synthesized in vivo or in vitro. Endogenous RNApolymerase of the cell may mediate transcription in vivo, or cloned RNApolymerase can be used for transcription in vivo or in vitro. Fortranscription from a transgene in vivo or an expression construct, aregulatory region (e.g., promoter, enhancer, silencer, splice donor andacceptor, polyadenylation) may be used to transcribe the RNA strand (orstrands). The RNA strands may or may not be polyadenylated; the RNAstrands may or may not be capable of being translated into a polypeptideby a cell's translational apparatus. RNA may be chemically orenzymatically synthesized by manual or automated reactions. The RNA maybe synthesized by a cellular RNA polymerase or a bacteriophage RNApolymerase (e.g., T3, T7, SP6). If synthesized chemically or by in vitroenzymatic synthesis, the RNA may be purified prior to introduction intothe cell. For example, RNA can be purified from a mixture by extractionwith a solvent or resin, precipitation, electrophoresis, chromatography,or a combination thereof. Alternatively, the RNA may be used with no ora minimum of purification to avoid losses due to sample processing. TheRNA may be dried for storage or dissolved in an aqueous solution. Thesolution may contain buffers or salts to promote annealing, and/orstabilization of the duplex strands.

In another aspect, the present invention provides pharmaceuticalcompositions comprising the polypeptide or GPBP silencers of this aspectof the invention or pharmaceutically acceptable salts thereof, and apharmaceutically acceptable carrier.

These peptides, or pharmaceutical compositions thereof, can be used inmethods for treating one or more of autoimmune conditions and a proteindeposit-mediated disorder, which comprise providing an amount effectiveof the polypeptides or GPBP silencers to a patient in need thereof totreat the autoimmune condition and/or a protein deposit-mediateddisorder. The terms “autoimmune condition” and “protein deposit-mediateddisorder” are as defined above.

As used herein, “treat” or “treating” means accomplishing one or more ofthe following: (a) reducing the severity of the disorder; (b) limitingor preventing development of symptoms characteristic of the disorder(s)being treated; (c) inhibiting worsening of symptoms characteristic ofthe disorder(s) being treated; (d) limiting or preventing recurrence ofthe disorder(s) in patients that have previously had the disorder(s);and (e) limiting or preventing recurrence of symptoms in patients thatwere previously symptomatic for the disorder(s).

In a further embodiment, the present invention provides methods forinhibiting GPBP activity, comprising administering to a patient in needthereof an amount effective to inhibit GPBP activity of one or morenovel polypeptides or silencers according to this aspect of theinvention. As used herein, the term “inhibiting” or “inhibit” means todecrease GPBP expression or activity, such as decreasing GPBP kinaseactivity.

The present invention further provides methods for treating one or moreof an autoimmune disorder and a protein deposit-mediated disordercomprising administering to a subject in need thereof an amounteffective to treat the disorder of a compound selected from the groupconsisting of staurosporine, Ca²⁺ CaM,1-[N,O-bis-(5-Isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine(KN62), and2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine(KN-93), or pharmaceutically acceptable salts thereof. The experimentalresults below demonstrate that each of these compounds is, either aloneor in combination with other compounds, an inhibitor of GPBP activity.

For administration, the polypeptides, nucleic acids, or other compoundsdisclosed above (hereinafter referred to collectively as “compounds”)are ordinarily combined with one or more adjuvants appropriate for theindicated route of administration. The compounds may be mixed withlactose, sucrose, starch powder, cellulose esters of alkanoic acids,stearic acid, talc, magnesium stearate, magnesium oxide, sodium andcalcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodiumalginate, polyvinylpyrrolidine, and/or polyvinyl alcohol, and tabletedor encapsulated for conventional administration. Alternatively, thecompounds of this invention may be dissolved in saline, water,polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidalsolutions, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil,tragacanth gum, and/or various buffers. Other adjuvants and modes ofadministration are well known in the pharmaceutical art. The carrier ordiluent may include time delay material, such as glyceryl monostearateor glyceryl distearate alone or with a wax, or other materials wellknown in the art.

The compounds of the invention can be administered as the sole activepharmaceutical agent, or they can be used in combination with one ormore other compounds useful for carrying out the methods of theinvention. When administered as a combination, the therapeutic agentscan be formulated as separate compositions that are given at the sametime or different times, or the therapeutic agents can be given as asingle composition.

The compounds may be made up in a solid form (including granules,powders or suppositories) or in a liquid form (e.g., solutions,suspensions, or emulsions). The compounds of the invention may beapplied in a variety of solutions and may be subjected to conventionalpharmaceutical operations such as sterilization and/or may containconventional adjuvants, such as preservatives, stabilizers, wettingagents, emulsifiers, buffers etc.

The compounds of the invention may be administered orally, topically,parenterally, by inhalation or spray or rectally in dosage unitformulations containing conventional non-toxic pharmaceuticallyacceptable carriers, adjuvants and vehicles. The term parenteral as usedherein includes percutaneous, subcutaneous, intravascular (e.g.,intravenous), intramuscular, or intrathecal injection or infusiontechniques and the like. In addition, there is provided a pharmaceuticalformulation comprising a compound of the invention and apharmaceutically acceptable carrier. One or more compounds of theinvention may be present in association with one or more non-toxicpharmaceutically acceptable carriers and/or diluents and/or adjuvants,and if desired other active ingredients. The pharmaceutical compositionscontaining compounds of the invention may be in a form suitable for oraluse, for example, as tablets, troches, lozenges, aqueous or oilysuspensions, dispersible powders or granules, emulsion, hard or softcapsules, or syrups or elixirs.

Compositions intended for oral use may be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavoringagents, coloring agents and preservative agents in order to providepalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients that aresuitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as calcium carbonate, sodium carbonate,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, corn starch, or alginic acid;binding agents, for example starch, gelatin or acacia, and lubricatingagents, for example magnesium stearate, stearic acid or talc. Thetablets may be uncoated or they may be coated by known techniques. Insome cases such coatings may be prepared by known techniques to delaydisintegration and absorption in the gastrointestinal tract and therebyprovide a sustained action over a longer period. For example, a timedelay material such as glyceryl monosterate or glyceryl distearate maybe employed.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents may beadded to provide palatable oral preparations. These compositions may bepreserved by the addition of an anti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

Pharmaceutical compositions of the invention may also be in the form ofoil-in-water emulsions. The oily phase may be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations may also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions may be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension may be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose any bland fixed oilmay be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The compounds and pharmaceutical compositions of the present inventionmay also be administered in the form of suppositories, e.g., for rectaladministration of the drug. These compositions can be prepared by mixingthe drug with a suitable non-irritating excipient that is solid atordinary temperatures but liquid at the rectal temperature and willtherefore melt in the rectum to release the drug. Such materials includecocoa butter and polyethylene glycols.

Compounds and pharmaceutical compositions of the present invention maybe administered parenterally in a sterile medium. The drug, depending onthe vehicle and concentration used, can either be suspended or dissolvedin the vehicle. Advantageously, adjuvants such as local anesthetics,preservatives and buffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.01 mg to about 50 mg perkilogram of body weight per day, and more preferably between 0.1 mg toabout 50 mg per kilogram of body weight per day, are useful in thetreatment of the above-indicated conditions. The amount of activeingredient that may be combined with the carrier materials to produce asingle dosage form will vary depending upon the host treated and theparticular mode of administration. Dosage unit forms will generallycontain between from about 1 mg to about 500 mg of an active ingredient.

Pharmaceutical compositions containing the compounds described hereinare administered to an individual in need thereof. In a preferredembodiment, the subject is a mammal; in a more preferred embodiment, thesubject is a human. In therapeutic applications, compositions areadministered in an amount sufficient to carry out the methods of theinvention. Amounts effective for these uses depend on factors including,but not limited to, the nature of the compound (specific activity,etc.), the route of administration, the stage and severity of thedisorder, the weight and general state of health of the subject, and thejudgment of the prescribing physician. The active compounds areeffective over a wide dosage range. However, it will be understood thatthe amount of the compound actually administered will be determined by aphysician, in the light of the above relevant circumstances. Therefore,the above dosage ranges are not intended to limit the scope of theinvention in any way.

The present invention may be better understood with reference to theaccompanying examples that are intended for purposes of illustrationonly and should not be construed to limit the scope of the invention, asdefined by the claims appended hereto.

EXAMPLES

Synthetic oligonucleotides. The following oligonucleotides and othersused for cDNA sequencing were synthesized by Amershan Biosiences oRoche:

ON-hmbGPBP-5c, (SEQ ID NO:58) 5′-CCTCCGAGCCCGACGAGTTC-3′ ON-dinb1, (SEQID NO:59) 5′-GACCGAAAGGGGCACGCAAC-3′; ON-GPBPΔ102, (SEQ ID NO:60)5′-AAAAAGAATTCGCATCGAGGGGGCTAAGTTCGG-3′; ON-GPBPΔ174, (SEQ ID NO:61)5′-AAAAAGAATTCGACGGCTGGAAGGGTAGGCT-3′; ON-GPBPΔ246, (SEQ ID NO:62)5′-AAAAAGAATTCTGTCAGGCGCGGCGGCGGCGC-3′; ON-GPBPΔ315, (SEQ ID NO:63)5′-GACGAATTCCCATCCCCCGACCCTTCACCC-3′; ON-GPBPΔ369, (SEQ ID NO:64)5′-AAAAAGAATTCGGAGCGGGGGCCGGTCTCCTGC-3′; ON-pU1, (SEQ ID NO:65)5′-ACGACTCACTATAGGGAGAC-3′; ON-pcDNAc, (SEQ ID NO:66)5′-CTCTAGCATTTAGGTGACAC-3′; ON-GPBPMet_(mut) (SEQ ID NO:67)5′-GGTTGTCGAGCCTCCGGATCGGATAATCAGAGC-3′; ON-PrP-F3, (SEQ ID NO:68)5′-GAGAATTCAGCAGTCATTATGGCGAACCTT-3′; ON-PrP-R1, (SEQ ID NO:69)5′-GAACTCGAGCCTTCCTCATCCCACTATCAGG-3′; ON-E/K-PrP-F6, (SEQ ID NO:70)5′-TATCACCCAGTACAAGAGGGAATCT-3′; ON-E/K-PrP-R6, (SEQ ID NO:71)5′-AGATTCCCTCTTGTACTGGGTGATA-3′; ON-E168R-F1, (SEQ ID NO:72)5′-CCCATGGATAGGTACAGCAACC-3′; ON-E168R-R1, (SEQ ID NO:73)5′-GGTTGCTGTACCTATCCATGGG-3′; ON-Q172R-F1, (SEQ ID NO:74)5′-GAGTACAGCAACAGGAACAACTTTG-3′; ON-Q172R-R1, (SEQ ID NO:75)5′-CAAAGTTGTTCCTGTTGCTGTACTC-3′; ON-R220A-F1, (SEQ ID NO:76)5′-CAGTACGAGGCGGAATCTCAGG-3′; ON-R220A-R1, (SEQ ID NO:77)5′-CCTGAGATTCCGCCTCGTACTG-3′; ON-R228A-F1, (SEQ ID NO:78)5′-TATTACCAGGCAGGATCGAGCAT-3′; ON-R228A-R1, (SEQ ID NO:79)5′-ATGCTCGATCCTGCCTGGTAATA

cDNA cloning and plasmid constructs for deletion mutants. To generatethe cDNA for the different GPBP deletion mutants, we performed PCR onpc-n4′ using one of the following synthetic oligonucleotidesON-GPBPΔ102, ON-GPBPΔ174, ON-GPBPΔ246, ON-GPBPΔ315, ON-GPBPΔ369 andON-pcDNAc. The resulting cDNAs were individually cloned in EcoRI ofpc-DNΔ3 (Invitrogen) to generate the pc-n4′A series. To obtainYFP-Flag-n4′ and YFP-n4′Δ102, the cDNAs in pc-Flag-n4′ and pc-n4′Δ102were EcoRI excised and cloned in-frame into pEYFP-C1 (Clontech).

The cloning identification and characterization of cDNA for bovine andmouse GPBP and GPBPΔ26 has been reported (WO 00/50607 and WO 02/061430).The 5′ UTR region for rat GPBP mRNA was obtained by standard reversetranscriptase-coupled-PCR using ON-hmbGPBP-5c and ON-dinb1 and total RNAwas extracted from cultured rat astrocytes provided by C. Guerri atFVIB, and subsequent nucleotide sequencing of PCR product.

The pc-n4′Met_(mut) construct was obtained by Transformer™Site-DirectedMutagenesis (Clontech) using pc-n4′ and ON-GPBPMet_(mut) followingmanufacturer's instructions.

We used human DNA extracted from blood and ON-PrP-F3 and ON-PrP-R1 toobtain a DNA that was subsequently cloned in EcoRI and XhoI of pc-DNΔ3(Invitrogen) to produce pc-PrP. To produce the derived mutants we used adouble-PCR approach using complementary oligonucleotides(ON-E/K-PrP-F6/ON-E/K-PrP-R6; ON-E168R-F1/ON-E168R-R1;ON-Q₁₇₂R-F1/ON-Q₁₇₂R—R1; ON-R220A-F1/ON-R220A-R1;ON-R228A-F1/ON-R228A-R1) that introduce the desired mutation andON-pcDNAc or ON-pU1, and pc-PrP as a template. The resulting DNAs weresimilarly cloned in pc-DNΔ3.

SimRNA production. Silencers were generated using pSilencer 2.1-U6 hygroplasmid (Ambion) following manufacturers recommendations. Theoligonucleotide pairs used were:

SiGPBP/Δ26-1: (SEQ ID NO:80)5′GATCCCACTACATTCATGGGTGGCATTCAAGAGATGCCACCCATGAAT GTAGTTTTTTTGGAAA-3′and (SEQ ID NO:81) 5′AGCTTTTCCAAAAAAACTACATTCATGGGTGGCATCTCTTGAATGCCACCCATGAATGTAGTGG-3′. SiGPBP/Δ26-2: (SEQ ID NO:82)5′GATCCCACAGAGTATGGCTGCAGAGTTCAAGAGACTCTGCAGCCATAC TCTGTTTTTTTGGAAA-3′and (SEQ ID NO:83) 5′AGCTTTTCCAAAAAAACAGAGTATGGCTGCAGAGTCTCTTGAACTCTGCAGCCATACTCTGTGG-3′; SiGPBP/Δ26-3: (SEQ ID NO:84)5′GATCCCGTACTTTGATGCCTGTGCTTTCAAGAGAAGCACAGGCATCAA AGTACTTTTTTGGAAA-3′and (SEQ ID NO:85) 5′AGCTTTTCCAAAAAAGTACTTTGATGCCTGTGCTTCTCTTGAAAGCACAGGCATCAAAGTACGG-3′; SiGPBP/Δ26-4: (SEQ ID NO:86)5′GATCCCAGGCGTCACAGGACATGAATTCAAGAGATTCATGTCCTGTGA CGCCTTTTTTTGGAAA-3′and (SEQ ID NO:87) 5′AGCTTTTCCAAAAAAAGGCGTCACAGGACATGAATCTCTTGAATTCATGTCCTGTGACGCCTGG-3′; SiGPBP: (SEQ ID NO:88)5′GATCCCGCCCTATAGTCGCTCTTCCTTCAAGAGAGGAAGAGCGACTAT AGGGCTTTTTTGGAAA-3′and (SEQ ID NO:89) 5′AGCTTTTCCAAAAAAGCCCTATAGTCGCTCTTCCTCTCTTGAAGGAAGAGCGACTATAGGGCGG-3′.

GPBP Expression in yeast and purification of recombinant protein.Recombinant FLAG-tagged human GPBP was essentially prepared as indicatedin Raya, A., Revert, F., Navarro, S., and Saus J (1999) J. Biol. Chem.274, 12642-12649. For light scattering purposes FLAG-affinity purifiedGPBP was further purified by FPLC on a Resource-Q column (AmershamBioscience) equilibrated with 20 mM Tris HCl pH 8 and eluted in a lineargradient of NaCl 0-1M established in the same buffer and the peakcontaining the material which eluted at ˜0.6 M NaCl was aliquot andstored at −80° C. until use.

Recombinant protein expression in cultured cells. The pcDNΔ3-basedcontructs containing the cDNA encoding the different human proteins ofinterest were used to transfect human 293 cells using standard calciumphosphate procedures in ProFection Mammalian Transfection System(Promega). 24-48 h after transfection cell lysates were used for Westernblot, precipitation or immunoprecipitation studies. Cells expressingGPBP or derived deletion mutants were collected on ice with 50 mM TrisHCl pH 7.4, 0.05% Triton X-100, 1 mM PMSF and 5 μg/ml leupeptin,disrupted by vortex and insoluble material discarded by centrifugationat 14.000 rpm in Eppendorf at 4° C. for 10 min and supernatant used forWestern blot analysis.

For other purposes after transfection and prior analysis cells wereincubated with GPBP modulators or lysosomal inhibitors.

Recombinant protein expression in a cell-free system. Approximately 1 μgof the pcDNΔ3-based construct was expressed in a coupledtranscription-translation system (Promega) following manufacturer'srecommendation and using ³⁵S-Methionine. The mixtures were analyzed bySDS-PAGE and a standard procedure for fluorography.

Subcellular fractioning and related studies. Rat liver subcellularfractionation was essentially performed as indicated in Aniento, F.,Roche, E., Cuervo, A. M., Knecht, E. (1993). J. Biol. Chem. 268,10463-10470. For some purposes lysosomal fractions (freshly preparedentire lysosomes) were dispersed in pure water and subjected to tenconsecutive cycles of freezing and thawing to alter lysosomal membraneintegrity (broken lysosomes). For other purposes, lysosomal fractionswere similarly disrupted in the presence of protease inhibitors (PMSF 2mM, leupeptin 0.2 mM and EDTA 2 mM) and subsequently centrifuged130,000×g for 10 min at room temp to separate the soluble lysosomalfraction (also called here lysosomal extract) from the non-solublefraction which, after rinse with 0.3 M saccharose in 10 mM MOPS pH 7.2,was used as the lysosomal membrane fraction.

To determine whether the GPBP immunoreactive polypeptides in lysosomal,microsomal and mitochondrial fractions represented cytoplasmiccomponents non-specifically bound to these organelles we subjected eachindividual fraction to five consecutive washes with 0.3 M saccharose in10 mM MOPS pH 7.2. Similar amounts of individual fractions representingeach wash were analyzed by Western blot using Mab6. For similarpurposes, mitochondrial and lysosomal fractions were treated withtrypsine at different concentrations for 1 h at room temperature,digestion stopped by adding soybean trypsin inhibitor and samplessimilarly analyzed. In these cases, Western blot was performed inparallel with Mab6 and either anti-catepsin D antibodies for lysosomalsamples or anti-carbamyl phosphate synthetase for mitochondrialfractions as degradation control of an integral component.

For still other purposes, entire or broken lysosomes (50 μg) wereincubated at 30° C. with 25 mM β-glycerol phosphate pH 7, 0.5 mM EGTA, 8mM Mg Cl₂, 5 mM MnCl₂, 1 mM DTT, y 0.132 μM [γ³²P]-ATP, maintainingsaccharose concentration to 0.25 M in a final volume of 50 μl. The timeof incubation was between 0 and 60 min and the phosphate transferreactions stopped by adding SDS-PAGE sample buffer and heating at 95° C.

Fluorescence microscopy studies. In a typical assay 20.000 cells wereseeded on glass slides and after 12 h the cells were rinsed with PBS(phosphate buffered saline) and fixed with methanol/acetone (50:50) for5 min at −20° C. Cells were brought to room temperature by rinsing withPBS and used for indirect immunofluorescence. Briefly, cells wereblocked for 45 min with PBS 3% BSA and then subsequently incubated forperiods of 45 min with the corresponding primary and secondaryantibodies. Finally, the slides were mounted for observation. A ZeissAxioskop 2 microscope was used for standard fluorescence microscopy andan ACAS 570 interactive laser cytometer using a pinhole size of 225 mmcorresponding to a 0.99 mm slice for confocal microscopy.

For other purposes, Cos-1 and HeLa cells were grown in 22 mm glasscoverslips and transient transfections were performed 24-36 hours afterseeding using SuperFect (Qiagen) or Fugene (Roche) transfectionreagents. 24-48 hours after transfection, cells were washed with HBSS(Hanks buffered salt solution) containing 5 mM glucose and 10 mM Hepes,pH 7.4. Coverslips were transferred to a microscopy chamber (Attofluor,Molecular Probes, The Netherlands) and cell fluorescence was imaged withan epifluorescence inverted microscope (DMIRE-2, Leica Microsystems,Germany) equipped with an oil immersion 40× objective (NA 1.25).Fluorescence was excited at 475 nm (YFP) using a monocromator (HamamatsuPhotonics, Japan) and emitted light collected by a CCD camera (Orca-ER,Hamamatsu Photonics, Japan). The emission filter (Omega Optical,Brattleboro, Vt., USA) was 535±13 nm and the beam splitter was 445DRLP.Images were acquired and analyzed using the Aquacosmos software(Hamamatsu Photonics, Japan).

Animal studies. NZW, male or female, 4-6 month-old were injectedintraperitoneally either with 1 μg/g of body weight of DAB-Am-4 and/orwith 20 μg/g of body weight of the Q_(2L) or Q_(2D) peptide. Theseproducts were administered in a volume of 500 μl of steril salinesolution 3 times per week, at alternate days, during 12 consecutiveweeks. Age-matched uninjected mice were used as controls. At the end ofthe experiment, mice were sacrificed and kidneys were fixed in 10%paraformaldehyde and processed for pathological studies. SimilarDAB-Am-4 treatment studies were performed on C57BL/6 animals for geneticbackground control.

Light-scattering studies. A 0.7 μM solution of bovine recombinantPrP^(C) (Prionics) in 20 mM Mes pH 6.5 buffer supplemented with 20 mMNaCl and 1 mM sodium citrate was placed in the measurement cell. After10 min FPLC-purified human recombinant GPBP was added from a stocksolution in TBS (Tris-buffered saline, 50 mM Tris-HCl pH 8, 150 mM NaCl)to rich a final concentration of 0.19 μM. In a second type of experimentthe protein initially placed in the measurement cell was GPBP andPrP^(C) was the added protein. For other purposes, GPBP solution wasplaced in the measurement cell and 10 min after inhibitory Q_(2L) ornon-active Q_(2Lr) (100 μM) or Q_(2D) (20 μM) were added, incubationcontinued for an additional 5-10 min period and PrP^(C) added. Lightscattering at 90° was recorded on a JASCO FP6500 spectrofluorimeter at500 nm as a function of time.

SDS-PAGE, Western and far Western studies. These studies wereessentially performed as indicated in Raya, A., Revert, F., Navarro, S.,and Saus J (1999) J. Biol. Chem. 274, 12642-12649 and Raya, A. et al.,(2000) J. Biol. Chem. 275, 40392-40399.

Yeast two-hybrid studies. Yeast two hybrid-studies to map interactivemotifs for GPBP self-aggregation were performed essentially as describedin Raya, A. et al., (2000) J. Biol. Chem. 275, 40392-40399 usingdifferent deletion mutants for GPBP obtained by standard DNA recombinanttechniques.

Precipitation and immunoprecipitation studies. After transfection ormodulator treatment, cells were washed once with ice-cold PBS, lysedwith 100-300 μl of lysis buffer (20 mM Tris-HCl pH 8, 100 mM NaCl, 0.5%NP-40 0.5%, sodium deoxycholate, 1 mM PMSF and leupeptin 10 μg/ml) andprotein concentration estimated using Bio-Rad protein assay and bovineserum albumin as standard. For precipitation studies, equal amounts ofprotein were brought to 501 with lysis buffer and centrifuged at16.000×g for 15 min at 4° C. Supernatants and pellets were analyzed byWestern blot. For immunoprecipitation studies lystes were pre-cleared at500×g for 5 min at 4° C. before protein quantification and equal amountsof protein were brought to 250 μl with lysis buffer. 5 volumes werediluted with TBS and incubated with anti-FLAG M2-Agarose Affinity Gel(Sigma) for 1 h at 4° C. with gentle agitation; beds were washed threetimes with TBS and used for Western blot analysis using biotin-labeledantibodies.

Cell cultures with GPBP modulators. One day after transfection (forPrP-expressing cells) or after seeding (for α3(IV) NC1-expressingcells), culture media were replaced with media (PrP-expressing cells) orserum-free media (α3(IV)NC1-expressing cells) containing GPBP modulatorsand cultures were extended for an additional 24 h (PrP-expressing cells)or 24-48 h (α3(IV)NC1-expressing cells). Cell lysates from PrPexpressing cells were used for Western blot, precipitation andimmunoprecipitation studies, and culture media from α3(IV)NC1 expressingcells for Western blot analysis. For α3(IV)NC1-expressing cells,synthetic peptides were used at 100-200 μM and organic compounds at 5-50μM. For PrP-expressing cells, Q_(2D) was used at 1-10 μM, DAB-Am-4 at1-5 μM and DAB-Am-32 at 0.25-1 μM.

In vitro phosphorylation. These studies were essentially performed asdescribed in Raya, A., Revert, F., Navarro, S., and Saus J (1999) J.Biol. Chem. 274, 12642-12649. Where indicated GPBP modulators were usedat 200 μM in a 10 min autophosphorylation reaction. Furtherautophosphorylation studies were performed using DAB-Am-4 and Q_(2D) andwe have determined that similar activation and inhibition effects wereobtained using decreasing concentrations up to 10 μM.

Histochemical and immunohistochemical on paraffin-embedded tissues.Immunohistochemical studies were essentially performed as indicated inRaya, A., Revert, F., Navarro, S., and Saus J (1999) J. Biol. Chem. 274,12642-12649 and Raya, A. et al., (2000) J. Biol. Chem. 275, 40392-40399.Hematoxylin/eosin and trichromic Mason staining on mice kidney sampleswere performed following standard procedures.

Antibody production. The production of chicken polyclonal antibodiesagainst GPBPpep1 recognizing GPBP and monoclonal antibodies againstGST-GPBP recognizing GPBP/GPBPΔ26 (Mab14) have been previously describedin Raya, A., Revert, F., Navarro, S., and Saus J (1999) J. Biol. Chem.274, 12642-12649 and Raya, A. et al., (2000) J. Biol. Chem. 275,40392-40399. Similar procedures were used for production of chickenpolyclonal antibodies against GPBPpep2 recognizing non-canonicalsequence of GPBP/GPBPΔ26 and monoclonal antibodies against GPBPpep1 onlyreacting GPBP (Mab6). For immunofluorescence and immunohistochemistrystudies we used polyclonal antibodies whereas monoclonals were used forWestern and far Western blot studies. The production andcharacterization of monoclonal antibodies against α3(IV)NC1 domain waspreviously reported (WO 02/061430). For some purposes antibodybiotinylation was performed as described in AntibodyArray™ InstructionManual from Hypromatrix.

Cell lines. The human cells lines used were HEK293 (ATCC), hTERT-RPE1and hTERTBJ1 (Clontech). The cell line used for α3(IV)NC1 expression wasobtained by stably transfecting HEK293 cells and its production has beenpreviously reported (WO 02/061430).

Other products: Synthetic peptides GPBPpep1,Ac-PYSRSSSMSSIDLVSASDDVHRFSSQ-NH2 (SEQ ID NO:46) and GPBPpep2,Ac-PRSARCQARRRRGGRTSS-NH2 (SEQ ID NO:36) were from Genosys. Syntheticpeptides Q₄ (Ac-EKTAGKPILF-OH) (SEQ ID NO:45),Q_(2LI)(Ac-ILATLSHCIELMVKR-NH2) (SEQ ID. NO:43),Q_(2L)(Ac-LATLSHCIELMVKR-NH2) (SEQ ID NO:90) Q_(2Lr)(Ac-VLMASLETLCRIHKI-NH2) (SEQ ID NO:92), Q_(2DI)(Ac-ILATLSHCIEL4VKR-NH2) (SEQ ID NO:43) and Q_(2D)(Ac-LATLSHCIELMVKR-NH2) (SEQ ID NO:90) were synthesized at the FVIB.Initial in vitro and ex vivo studies were performed using Q_(2L) andQ_(2DI) however further synthesis and uses were performed in absence offirst isoleucine and we synthesized Q_(2L) and Q_(2D) peptides that showsimilar activity both in vitro and ex vivo but were more soluble andused for animal studies. Antibodies for co-localization wereanti-catepsin D from Santa Cruz Biotechnology; anti-Golgin-91 (CDF4) andanti-human E2 subunit of pyruvate dehydrogenase from Molecular Probes.Anti-GAPDH and anti-carbamoyl phosphate synthetase were kindly providedby E. Knecht and J. Cervera at FVIB. GPpep1bov(Ac-KGKPGDTGPPAAGAVMRGFVFT-NH2) (SEQ ID NO:93) was synthesized byDiverDrugs and antibodies specific provided by Billy G. Hudson. Aβ₁₋₄₂and FLAG peptides and the corresponding specific antibodies were fromSigma. All the conjugates used except anti-mouse Ig peroxidase (Promega)were from Sigma. Recombinant bovine PrP was from Prionics. PrP-specificantibodies were from Chemicon (clone 3F4) or from Santa CruzBiotechnology (C-20). Rat cerebellar neuronal extracts were preparedessentially as described in Miñana M D, Montoliu C, Llansola M, GrisolíaS, Felipo V. (1998) Neuropharmacology 137; 847-857 were provided by V.Felipo at FVIB. SiGFP, an mRNA silencer for green fluorescence proteinwas from Ambion.

Results

Identification of a 91-kDa isoform of GPBP (91 kDa GPBP) as noncanonical mRNA translation start site product. We have made theobservation that the 5′ untranslatable region (5′UTR) of the mRNA ofhuman GPBP contains an upstream open reading frame (ORF) of 130 residueswith an in-frame stop codon at the beginning (See WO 00/50607). In vitroor ex vivo translation of the n4′ mRNA (n4′) resulted in the expressionof two molecular species, one consistent with canonical translation atiMet displaying a molecular mass of ˜77-kDa (77 kDa GPBP) and other withan apparent higher molecular mass (˜91-kDa). To investigate the natureof the 91-kDa molecular species (91 kDa GPBP), a cDNA representing anmRNA with no 5′UTR was obtained and similarly expressed. The expressionof this mRNA mutant resulted in a single protein of ˜77-kDa (77 kDaGPBP), indicating that the existence of 91-kDa GPBP depends onnon-canonical translation of the ORF at the 5′UTR. A cDNA representing aMet to Gly mRNA mutant for translation initiation expressed only the91-kDa molecular species (FIG. 1), indicating that the 91-kDa GPBP isexpressed from a non-canonical translation start site located 5′ fromthe codon encoding the canonical Met initiation codon. Similar 91 kDaGPBP isoforms were shown to be present in mouse and rat cells, and arepredicted to be expressed from GPBP mRNA in bovine cells.

The 91-kDa GPBP isoform results from previously unrecognized mRNAtranslation mechanism. To explore the mechanism underlying expression of91-kDa GPBP, we generated mutants representing truncated versions of themRNA at the 5′UTR and performed recombinant expression in a cell-freesystem (in vitro) or in cultured human cells (ex vivo) (FIG. 2).

Whereas all the deletion mutants expressed the canonical polypeptide of77-kDa GPBP, 91-kDa GPBP was only expressed from the complete mRNA (n4′)and from a mutant which is devoid of the 5′ 102 nucleotides (Δ102).Additional 5′ deletions failed to abolish non-canonical translationinitiation and caused a gradual reduction in the size of thenon-canonical product (FIG. 2), suggesting that there are multiplenon-canonical translation start sites displaying 5′ to 3′ hierarchy.

The relative expression of the two polypeptides in cell-free system (invitro) sharply contrasted with the levels of these two polypeptides whenthe mRNA was expressed inside the cell, in which case 77-kDa GPBPisoform was significantly more abundant.

In cells and tissues, the expression of GPBP mainly depends on thenon-conventional translation of the corresponding mRNA. In a firstattempt to investigate the significance of our findings we comparedrecombinant and endogenous expression of GPBP in cultured human 293cells (FIG. 3). As expected monoclonal antibodies specificallyrecognizing GPBP (Mab6) reacted with the two recombinant molecularspecies being expressed from cDNA in cell extracts deriving fromtransfected cells (91 kDa GPBP and 77 kDa GPBP) (lane 1). Cell extractsderived from non-transfected cells expressed several reactivepolypeptides (lane 2), one co-migrating with recombinant 91-kDa GPBP andother polypeptides of higher and lower molecular mass (120-, 47- and32-kDa), none of which displayed the molecular mass of the canonicalrecombinant polypeptide (compare lanes 1 and 2). In some studies thepresence of an additional 60-kDa polypeptide also was evident. Thespecificity of the multiple reactivity displayed by Mab6 was confirmedby full inhibiting antibody binding in the presence of GPBPpep1, asynthetic peptide representing the GPBP exclusive 26-residues used inMab6 production (lane 3). These findings suggest that at the steadystate of the cell canonical translation product is virtually absentwhereas non-canonical 91-kDa GPBP product is comparatively moreabundant. Furthermore, we identified an additional major GPBP isoformsof 120-kDa along with minor lower molecular mass GPBP isoforms of60-kDa, 47-kDa and 32-kDa.

To further investigate the nature of the immunoreactive polypeptides, wegenerated a number of GPBP mRNA silencers and assayed their capacity toinhibit endogenous expression of GPBP related polypeptides (FIG. 4). Allindividual mRNA silencers displaying the capacity to inhibit recombinantGPBP expression (not shown) also negatively impacted endogenousexpression of 91 kDa GPBP and 120-kDa GPBP. The consequences onexpression of GPBP polypeptides of lower molecular mass variedsubstantially between silencers. Thus, silencers that were moreefficient reducing 91 kDa GPBP and 120-kDa GPBP promoted the expressionof 60 kDa GPBP and reduced in an opposite but coordinated manner theexpression of 47-kDa GPBP. All these data suggest that the expression of91 kDa GPBP and 120-kDa GPBP depends more on mRNA translation than theexpression of GPBP isoforms of lower molecular mass, which depend on acomplex degradation program operating on the primary products andinvolving a positive feedback of the primary products in the proteolyticstep from 60- to 47-kDa. Our findings support that all the polypeptidesreactive with Mab6 indeed are GPBP related products, and suggest that atthe cellular steady state GPBP expression depends more on non-canonicaltranslation than on canonical translation of the mRNA.

We have extensively studied the expression of GPBP in multiple humantissue extracts and found an expression pattern that in general wassimilar to that found with cultured cells with the exception of a humanstriated muscle sample in which case we identified a minor reactivepolypeptide of 77-kDa. In this case it remained unclear whether the77-kDa polypeptide represented a canonical translation event orrepresented a proteolytical intermediate deriving from non-canonical91-kDa GPBP (see below). Although the structural relationship betweenthe 91-kDa and polypeptides of higher and lower molecular mass remainsto be determined, the evidence suggest that non-canonical translation ismore relevant in vivo than canonical. Immunochemical studies performedon paraffin embedded human tissues revealed that the immunostainingpatterns obtained using antibodies that recognize canonical andnon-canonical GPBP isoforms are virtually identical to those obtainedwith antibodies only reacting with non-canonical GPBP products.

In cultured cells, GPBP shows multiple subcellular localizationincluding a prominent presence at the endosomal/lysosomal compartmentimmunohistochemical studies support that non-canonical GPBP isoformsdisplay a broad subcellular localization including extracellular matrix,plasma membrane, cytosol (homogenous, fibillar and granular) and nucleus(WO 00/50607). An analysis for prediction of subcellular localizationsupports the multiple localization for non-canonical versus canonicalproducts (see below), despite the fact that many of these destinies arenon-compatible using conventional protein sorting routes. Consequently,we have explored GPBP subcellular localization using conventionalimmunofluorescence and confocal microscopy in cultured human cells.Indirect immunofluorescence studies on human RPE and BJ1 cellsrespectively representing epithelial and fibroblastic type of cellsimmortalized by telomerase (Clontech) revealed two principal cellularexpression patterns for GPBP. Most of the cells express GPBP at thecytosol in a diffuse and fibrillar manner, with a remarkable expressionof the protein at the nuclear membrane and perhaps presence of theprotein in the nuclear environment. A limited number of cells showabundant GPBP expression at granular structures that distribute in theperinuclear region. The percentage of cells expressing the intensegranular pattern varied between 10-30% and was more abundant in BJ1 thanin RPE cells.

We have explored the cells in which GPBP is over expressed at definedgranules to identify their subcellular nature. We performed conventionaldouble indirect immunofluorescence and confocal microscopy usingvalidated immunological probes for secondary lysosomes, Golgi apparatusor mitochondria. These studies revealed that GPBP shows a preferentiallocalization at the lysosomes and a more limited but significantpresence in Golgi apparatus and mitochondria. We have also performedfluorescence studies directed to address the intracellular distributionof proteins representing canonical or non-canonical GPBP primaryproducts fused to yellow fluorescence protein. These studies revealed amajor granular distribution for the non-canonical GPBP whereas thecanonical GPBP appeared to be mainly diffuse cytosolic.

All these results suggest that the endosomal/lysosomal compartment is aprincipal subcellular destiny for non-canonical GPBP.

The 91-kDa GPBP is the precursor of multiple related polypeptidesincluding lysosomal 44-47-kDa isoforms. To investigate subcellularlocalization of GPBP in tissues we have used rat liver, a validated andreliable model for cell subfractioning. From the correspondinghomogenates we prepared cytosolic, mitochondrial, microsomal andlysosomal fractions and assessed the presence of GPBP by Western blot(FIG. 5). The antibodies reacted with multiple polypeptides, whichdisplayed 120-, 91-, 77-, 60-, 44-47, and 32-kDa. The distribution ofreactive polypeptides among cellular fractions greatly varied. Thepolypeptides of higher molecular mass, (120-, 91-, and 77-kDa) werefound preferentially in microsomes, the polypeptide of 60-kDa was mainlyfound in cytosol and mitochondria with traces in microsomes, whereas44-47-kDa polypeptides were essentially lysosomal. Finally, the 32-kDapolypeptide was the most widely distributed being found in everyfraction, followed by the 60-kDa polypeptide that was found in allfractions except in lysosomes. Extensive washing or trypsin treatment ofeither mitochondrial or lysosomal fractions resulted in no significantreduction in the content of immunoreactive peptides, suggesting thatthese polypeptides are integral components of these cell compartments.

All our findings suggest that the primary products of GPBP mRNAtranslation are subjected to a complex intracellular processing coupledto subcellular localization. Immunofluorescence studies suggest that theendosomal/lysosomal compartment is among the most prominent destinationsfor GPBP. This compartment is also of major interest inantibody-mediated autoimmune pathogenesis and in tissue degeneration,since is actively implicated in the production of both non-tolerizedpeptides and in protein deposition. To investigate the presence of GPBPin this compartment, we isolated lysosomes from the liver of untreatedor leupeptin-treated rats, and the presence of GPBP was investigated byWestern blot analysis using specific monoclonal antibodies (FIG. 6).Lysosomes from untreated animals contained major reactive polypeptidesof 44-47-kDa and a minor polypeptide of 32-kDa. Treatment with leupeptinsubstantially changed the immunoreactive pattern and thus polypeptidesof higher molecular mass (91- and 60-kDa), virtually undetectable inuntreated lysosomes were the most abundant whereas the 44-47-kDapolypeptides significantly diminished. Although in most of the casesleupeptin treatment did not change the level of 32-kDa there wereexamples in which we found increased levels of this polypeptide, and insome other cases treatment was associated with detection of 77-kDa and120-kDa polypeptides. These data suggest that the 44-47-kDa polypeptidesare integral components of the lysosome that derive from limitedleupeptin-sensitive proteolysis of a 91-kDa precursor through a majorintermediate of 60-kDa. Furthermore, the non-reduced or moderatedaugmented expression of 32-kDa polypeptide associated with leupeptintreatment, suggests that the 91-kDa polypeptide is also subjected to asecond degradation process which is leupeptin-insensitive.

To further localize and to investigate the relationship among thedifferent GPBP-related polypeptides, we isolated matrix and membranesfrom lysosomes of treated or untreated rats and the correspondingextracts were similarly analyzed. Western blot studies on untreatedlysosomal fractions revealed that the major 44-47-kDa polypeptides arelocated at the lumen and with the exception of the 60-kDa polypeptide,which appeared in some preparations equally distributed between matrixand membrane fractions, the polypeptides other than the 44-47 kDa werefound to be membrane-associated components. These findings, in additionto further confirming that the 44′-47-kDa GPBP-related polypeptides areintegral components of the lysosomes provide some insights on themechanism of their production. Without being limited to a specificmechanism, we propose that the 91-kDa form exists exclusively attachedto the inner face of the lysosomal membrane where it undergoes limitedleupeptin-sensitive or leupeptin-insensitive proteolysis to yield the44-47-kDa or the 32-kDa polypeptides respectively. Leupeptin-sensitiveprocessing is predicted to occur in two principal steps, one with thepolypeptide being attached to the membrane yielding a 60-kDa product,and the other requiring release of the 60-kDa intermediate from themembrane and yielding a final products of soluble 44-47-kDapolypeptides. Leupeptin-insensitive processing appears to occur howeveronly on a membrane-bound 91-kDa polypeptide to generate a final productof 32-kDa still bound to the membrane. It remains to be determinedwhether the 77-kDa polypeptide found in some leupeptin-treated lysosomesrepresents a proteolytic intermediate or the canonical translationprimary product that also enters into this compartment.

All our findings suggest that the 44-47-kDa polypeptides are lysosomalisoforms of GPBP, which mainly derive from non-canonical 91-kDa GPBP.These studies also reveal that, with the exception of the 120-kDapolypeptide, lysosomes contain the enzymatic resources to generate allthe GPBP isoforms found in tissue and cell homogenates. This wasspecifically confirmed by coupling recombinant expression of the mRNA ina cell-free system coupled to limited proteolysis using rat liverlysosomal extracts (FIG. 7). Recombinant expression of mRNA coupled tolimited proteolysis produced polypeptides of 77-, 60-, 47-, 44- and32-kDa revealed that lysosomal proteolysis of primary translationproducts accounts for all the related polypeptides of lower molecularmass. Similar results were obtained when the proteolytic assays wereperformed using individual recombinant product (91- or 77-kDa).

Identification of phosphate transfer activity in isolated intactlysosomes and phosphorylation of the 44-kDa GPBP form. The above datasuggest that the 44-47 kDa polypeptides are the isoforms of GPBP in thelysosome. We assessed the ability of these polypeptides to transferphosphates by incubating intact or broken lysosomes with [γ³²P]-ATP andfurther analyzing the mixtures by SDS-PAGE and autoradiography (FIG.8A). Untreated, intact lysosomes incubated with isotonic buffer at pH 7efficiently incorporated ³²P at components of 44-47-, 34- and 3.2 to15-kDa, whereas lysosomal disruption greatly impaired labeling of thesematerials. To determine the relationship between molecular species thatincorporated ³²P and GPBP, a protein kinase with a prominent capacity toundergo autophosphorylation, we combined Western blot andautoradiography of SDS-PAGE analysis of phosphorylation mixturesrepresenting control or leupeptin-treated lysosomes (FIG. 8B). Thesestudies revealed the presence of labeled components co-migrating with44-47-kDa polypeptides in control lysosomes in addition to a labeledcomponent of lower molecular mass (˜34-kDa) not associated to anyimmunoreactive species. In general, we observed a fast initial labelingand a gradual reduction in ³²P-labeling with the time of incubation. Thestudies on leupeptin-treated lysosomes revealed a more efficient initial³²P labeling of virtually all the components previously identified incontrol lysosomes. However, in treated lysosomes the gradual reductionin ³²P-labeling of the 44-47-kDa components was accompanied by a graduallabeling of a component that co-migrated with 60-kDa GPBP isoform.Similarly, labeling reduction of non-immunoreactive 34-kDa material wasaccompanied by increased labeling of a component with higher molecularmass (˜38-kDa) that was also not detectable by monoclonal antibodies.These findings suggest that lysosomal GPBP isoforms are either thetarget of an unknown protein kinase therein or, more likely, thelabeling of the GPBP isoforms is the result of an autophosphorylationevent. Accordingly, a C-terminal deletion mutant of GPBP withapproximately 44-kDa molecular mass (residues 1-299 of GPBP) displayedgreater auto-phosphorylation activity at pH 5 than full sized GPBP,suggesting that lysosomal 44-47-kDa are GPBP isoforms more efficientthan 91- and 77-kDa primary products to operate inside the lysosome. Inany event, the demonstration of ³²P incorporation at the 60- andt44-47-kDa polypeptides provides, to our knowledge, the first evidencefor an intrinsic protein kinase activity in lysosomes and points to44-47-kDa GPBP as the first protein kinase operating inside this cellcompartment.

The conformational isomerization of the α3(IV)NC1 domain mainly occursat the endosomal/lysosomal compartment and depends on GPBP. Recombinantexpression of the α3(IV)NC1 domain in human 293 cells results in thesynthesis and secretion of multiple polypeptides ranging in size between22-27-kDa (WO 00/50607 and WO 02/061430). Reduction of disulfide bondsresults in a major single molecular species of 29-kDa and multiplederived proteolytic products of lower molecular mass, suggesting thatthe multiple polypeptides are conformational isoforms (conformers)maintained and stabilized by disulfide bonds that undergo limitedproteolysis (FIG. 9A). To explore the cell compartment at which theconformational diversification of the α3(IV)NC1 domain occurs, the cellswere cultured in the presence of NH₄Cl or leupeptin, lysosomotropicagents that increase the pH and inhibit cysteine proteases respectively.The presence of NH₄Cl reduced conformer production whereas leupeptininhibited the presence of proteolytic products, suggesting thatconformational diversification of the α3(IV)NC1 domain mainly occurs atthe endosomal/lysosomal compartment. To explore the role of GPBP inα3(IV)NC1 conformer production, compounds with the capacity to modulateGPBP kinase activity in vitro (see below) were used to regulate thecorresponding cellular conformer production (FIG. 9B). When a positiveGPBP modulator (DAB-Am4) was added to the culture medium an efficientincrease in conformer production occurred. In contrast, when thecompound added was a negative GPBP modulator (Q₂ or Q₄), a reduction inconformer production occurred. All these findings suggest that lysosomalGPBP isoforms are actively involved in the conformational isomerizationof the α3(IV)NC1 domain and consequently, the assembly of aberrantα3(IV)NC1 conformers mediating Goodpasture autoantibody production isexpected to be a lysosomal event. DAB-Am-4 is a branched polyamine andthese compounds have been shown to accumulate in secondary lysosomes(Supattapone S et al. J. Virol (2001) 75, 3453-3461).Fluorescein-labeled Q₂ showed a granular cell distribution with broadco-localization with GPBP as determined by indirect immunofluorescenceapproaches on cultured cells. D-amino acid version of Q₂, known to bemore refractory to degradation, was significantly more effectiveinhibiting GPBP cell conformer production.

GPBP, autoimmunity and tissue degeneration. Several lines of evidencesupport the idea that GPBP is involved in the pathogenesis of otherautoimmune diseases: 1) GPBP is preferentially expressed in cells andtissues that are targets of common autoimmune responses; 2) GPBP bindsto and phosphorylates other human autoantigens; and 3) Biochemical andimmunohistochemical studies show increased levels of GPBP expression intissues undergoing an autoimmune attack, including cutaneous lupuserythematosus (WO 00/50607) and more recently in cutaneous lesions ofpatients undergoing systemic lupus erythematosus (SLE).

The autoimmunity response in SLE is due to the involvement of bothgenetic and environmental factors. New Zealand White (NZW) mice, whichdo not develop autoimmunity, carry a genetic background that promotesSLE when bred with other mice strains, such as New Zealand Black (NZB).The genetic predisposition of NZW to undergo SLE, and more specificallyrenal lupus (autoimmune glomerulonephritis), has not been associatedwith any specific gene(s). In an attempt to relate GPBP with thisgenetic background, we have performed histological andimmunohistochemical studies to address the expression of GPBP in therenal glomerulus of NZW. Our studies suggest that these mice do notundergo a frank autoimmune response. However, 7-9 months after birththey develop a degenerative glomerulopathy that cause glomerulosclerosisand end-stage renal disease (ESRD) with a premature death at 13-14 monthof age. Morphologically, this nephropathy presents an evolution withseveral histological stages. Stage 1, characterized by minimal changesconsisting of slight cell proliferation (endothelial and/or mesangial)and light thickening of mesangium. Stage 2, the cell proliferation ismoderate and there are collagenous-like deposits at the mesangium whichstain with aniline blue, and other deposits of hyaline nature in thesubendothelial space that stain with acid fuchsin. Stage 3,characterized by an intense cell proliferation predominantly mesangialwith extensive protein deposits that invade subendothelial space (Stage3a), or endothelial with extensive nodular deposits that invade themesangium (Stage 3b). At this stage the more characteristichistochemical image is the presence of fuchsinophilic hyaline depositssurrounded by protein deposits of collagenous-like nature. Stage 4, inthis stage the glomeruli undergo sclerosis likely as a consequence ofscar organization of the collagenous deposits. When sclerosis resultsfrom deposits primarily mesangial this is more diffuse and homogeneous(Stage 4a) than when scar results from reactive fibrosis againstsubendothelial deposits, in which case this is more nodular andlaminated (Stage 4b).

Immunochemical studies performed to address the presence of GPBPrevealed that, in contrast to what has been previously described forcontrol mice kidneys (BALB/c and C57BL/6) (WO 00/50607), the NZW kidneysshow from moderate to abundant expression of GPBP in tubules and in theinterstitial spaces, without significant expression in the glomeruli.However, as glomerular degeneration starts and develops, we detectedGPBP expression at the subendothelial space in intimate association withthe fuchsinophilic subendothelial material. As the disease progressthrough Stages 3b and 4b, the expression of GPBP increasessubstantially.

Furthermore, in an attempt to relate the degenerative process and theproduction of autoantibodies, we have performed studies to address thepresence of immunoglobulin associated with material deposited in theglomerulus of NZW. These studies revealed the presence of lineardeposits of immunoglobulins in peripheral capillary loops in a number ofglomeruli that varied among individuals (focal and segmentarydistribution). As the degenerative process evolved, the number ofglomeruli showing linear deposits of immunoglobulins decreases,suggesting that these deposits are a marker of the glomerular structureswhich are going to undergo degeneration. Consistently, thesubendothelial deposits with nodular pattern characteristic of theStages 3b and 4b showed a high immunoglobulin content.

Finally, in an attempt to determine the nature of the proteinaceousmaterial deposited, we performed histochemical studies using Congo redand thioflavin T, compounds that become adsorbed to the protein depositsof amyloid nature and induce birefringence yellow-green of the polarizedlight or emit fluorescence, respectively. These studies revealed thatthe material, which is deposited in the subendothelial space as well asthe collagenous material at the mesangium adsorbed these two compounds.However, whereas thioflavin T was excitable and emitted fluorescence,the adsorbed Congo red was unable to induce birefringence to thepolarized light. These results indicate that the material depositedshares some structural features with amyloid matter (para-amyloid oramyloid-like).

Our studies suggest that the glomerular degeneration of NZW is primarilycaused by an alteration in the folding of certain proteins, which causeaggregation and deposit formation at the subendothelial space(fuchsinophilic deposits), and at the mesangium (aniline blue stainedmaterial). A reactive fibrosis against these deposits is likely thecause of glomerular sclerosis (end-stage renal disease, ESRD). Theprotein deposits although different than amyloid matter, share with itsome structural features. As previously described, we have not found afrank autoimmune response in NZW. However, the presence ofimmunoglobulins intimately associated with the subendothelial depositssuggest that, as in Goodpasture disease, aberrant conformers induceautoantibody production.

In light of all these findings, we suggest that an aberrant expressionof GPBP is part of the genetic background which predispose NZW mice toundergo tissue degeneration (deposition of proteins) and autoantibodyproduction (autoimmune response). Furthermore, the coordinated increaseof protein deposits, GPBP, and immunoglobulins at the subendothelialspace suggest that the three processes are related.

GPBP is a Molecular Target for Treating Diseases Mediated byAmyloid-Like Matter.

In an attempt to establish the causal relationship between GPBP activityand the formation of protein deposits in NZW, we have identifiedmodulators of the activity of GPBP and we administered them to thesemice.

Branched polymanines (dendrimers) are chemical structures with a largenumber of peripheral reactive amines which are commonly used to besubstituted by one or more chemical groups to increase their presence atthe molecular surface and thus enhance their biological/therapeuticactivity. We have found that branched polyamine of first generation[Sigma product numbers 46, 069-9: polypropylenimine tetraamine dendrimer(DAB-Am-4)] is a potent activator of GPBP kinase activity in vitro andα3(IV)NC1 conformer production in cultured cells (see above). Afterperforming toxicity assays in mice, we administered non-toxic doses ofDAB-Am-4 to 4-6 month-old NZW mice, and we studied its consequences inthe progression of the glomerulopathy. These studies revealed thatDAB-Am-4 caused an acceleration of the degenerative process, resultingin premature glomerulosclerosis at 7-9 month of age. Whereas in thenatural progression of the disease, the morphological pattern morefrequently found was that through Stages 3a and 4a, the treatmentinduces almost constantly a progression through Stages 3b and 4b withabundant presence of GPBP intimately associated with protein deposits.These data suggest that an augmented activity of GPBP is causallyrelated with the progression of the degenerative process towardssclerosis and ESRD. In trials on control mice (C57BL/6) we have notobserved histological changes of relevance due to administration ofDAB-Am-4, suggesting that the capacity for DAB-Am-4 to induce glomerularsclerosis depends mainly on the NZW genetic background which possiblyinvolves aberrant activation/expression of GPBP.

To verify that an induction of GPBP in the genetic context of NZW isresponsible for the degenerative process, NZW mice were treated withDAB-Am-4 or with DAB-Am-4 and Q₂, a synthetic peptide (LATLSHCIELMVKR)(SEQ ID NO:90) that encompasses a motif of GPBP for self-interaction intwo-hybrid studies, and thus suspected to be critical for GPBPaggregation, that efficiently inhibits GPBP kinase activity in vitro andα3(IV)NC1 conformer production in cultured cells (see above and belowsections). The treatment with Q₂ sharply reduced the material depositedin the glomerulus of NZW of suspected collagenous nature although it wasshown to be unable to reduce the presence of fuchsinophilic material atthe subendothelial space. A D-amino acid version of Q₂ was significantlymore effective than the L-amino acid version consistent with its morepotent inhibitory activity on GPBP kinase activity in vitro and oncellular conformer production.

One way to interpret these findings is that Q₂ efficiently blocksprogression from Stage 3b to 4b during disease induction by DAB-Am-4. Inother words, the presence of abundant fuchsinophilic material in Q₂treated mice is suspected to be caused by the lack of fibrotic reactionwhich substitutes or masks fuchsinophilic material during diseaseprogression. This results in glomeruli virtually devoid of fibroticreaction that causes ESRD.

When we assessed thioflavin T or Congo red staining, we found that thematerial which stains with acid fuchsin, contrary to the homologousmaterial at Stage 3b in natural disease or in DAB-Am-4 induced disease,did not adsorb either compound, suggesting that by inhibiting GPBP, Q₂efficiently inhibited amyloid-like matter formation in NZW. Finally,this specific effect on protein deposit structure could be responsiblefor an attenuated fibrotic reaction, and the lack of progression towardsglomerular sclerosis and ESRD.

Identification of multiple compounds that modulate GPBP kinase activityin vitro and α3(IV)NC1 conformer production in culture cells. Todetermine the role of GPBP in the conformational diversification of theα3(IV)NC1 domain, we have first identified and characterized differentmodulators of kinase activity of GPBP in vitro, and later we have usedthem to modulate conformer production in cultured cells.

We have reported that GPBP self-interacts and that aggregation regulateskinase activity (WO 00/50607). By combining a yeast two-hybrid systemand cDNA deletion mutants of GPBP, we have identified a five-residue(SHCIE) (SEQ ID NO:39) and a ten residue (EKTAGKPILF) (SEQ ID NO:45)motifs in the GPBP amino acid sequence that are critical forself-interaction. A synthetic peptide representing the five-residuemotif and flanking regions (LATLSHCIELMVKR, called here Q₂) (SEQ IDNO:90) efficiently inhibited GPBP autophosphorylation, whereas asynthetic peptide representing the ten-residue motif (EKTAGKPILF, calledhere Q₄) (SEQ ID NO:45) inhibited GPBP autophosphorylation in a morelimited manner. Furthermore, when these peptides where separately addedto the culture media of cells expressing α3(IV)NC1, Q₂ was moreeffective inhibiting cell conformer production than Q₄ which had a morelimited inhibitory effect (see above). A D-amino acid version of Q₂(Q_(2D)) was more efficient inhibiting GPBP autophosphorylation andconformer cell production than the corresponding L-amino acid version(Q_(2L)).

We have also assayed a number of protein kinase inhibitors (CalbiochemCat No 539572) and found that staurosporine (a broad range Ser/Thrkinase inhibitor) and KN93,2-[N-(2-hydroxyethyl)-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine[an inhibitor highly specific for Ca²⁺ Calmodulin-dependent proteinkinase II (CaM kinase II)], efficiently impaired GPBP kinase activity invitro. KN62,1-[N,O-bis-(5-Isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine[another specific inhibitor for CaM kinase II (Sigma Cat No I2142)],inhibited GPBP kinase activity in vitro to a larger extent than KN93,which in contrast is known to be a more potent inhibitor than KN62 forCaM kinase II. These organic compounds are thought to inhibit CaM kinaseII by interfering with the binding of the Ca²⁺ Calmodulin activatorcomplex to the kinase. In vitro studies consistently revealed thatcalmodulin inhibited GPBP kinase activity in a Ca²⁺ dependent manner,suggesting that GPBP contains a binding site for Ca²⁺ Calmodulin similarto that of CaM kinase II, although the consequences of binding on kinaseactivity were different in each case. Finally, when KN62 and KN93 wereseparately added to the culture media of α3(IV)NC1-secreting cells, wefound that these compounds reduced cell conformer production. Thesefindings, in addition to identify potential compounds for modulatingGPBP kinase activity and α3(IV)NC1 cell conformer production, uncoverimportant features of allosteric regulation of GPBP, and reveal thatGPBP has catalytic features that are similar to CaM kinase II. CaMkinase II also requires self-aggregation to be functional, and Q₂efficiently inhibited CaM kinase II, suggesting that the interactingmotifs of these two kinases are structurally related. Consistent withthis idea aggregatable CaM kinase II contains a highly homologousfive-residues motif, SHCIQ (SEQ ID NO:40), not present innon-aggregatable CaM kinases.

Although Q₄ appeared to be a poor inhibitor under experimentalconditions in which Q₂ efficiently inhibited GPBP kinase activity, thispeptide showed synergistic inhibitory effect on GPBP kinase activitywith KN62 (+++), Ca²⁺ CaM (++) and Q₂ (+). Similarly we found inhibitorysynergism on GPBP kinase activity when combining KN62 and Ca²⁺ CaM(+++), whereas no cooperative inhibitory effect was observable whencombining Q₂ and Ca²⁺ CaM or Q₂ and KN62.

In contrast to the positive regulatory effect of DAB-Am-4, fourthgeneration of branched polyamines (DAB-Am-32) (Sigma Chemical Co.product number 46, 908-4) efficiently inhibited GPBP protein kinaseactivity. This compound has been shown to accumulate in the lysosomesand exert a curative effect on prion infected cultured cells(Supattapone S. et al. J. Virol (2001) 75, 3453-3461).

These data, in addition to provide experimental evidence for the role ofGPBP in the conformational isomerization of the α3(IV)NC1 domain invitro and ex vivo, report the first repertoire of lead compounds to beuse for treatment of autoimmune diseases and protein deposit-mediateddisorders.

GPBP is a potential molecular target for treating amyloidosis. GPBPrepresents the first example of a molecular enzyme showing kinase andpolypeptide conformation isomerase activity (WO 00/50607 and WO02/061430). We have shown that a 91-kDa isoform of GPBP resulting fromnon-canonical mRNA translation enters the lysosome and undergoesprocessing to yield a 44-47-kDa product, which in turn represents a GPBPisoforms that are integral components of this cell compartment. Theevidence suggests that in this acidic environment, GPBP can transferphosphate and therefore conceivably could also catalyze conformationalisomerization reactions.

An important number of human degenerative diseases, includingAlzheimer's and prion diseases, are mediated by the presence ofaggregates (amyloid matter) made of non-soluble conformational versionsof specific cellular components (Aβ₁₋₄₂ and PrP^(Sc), respectively). Theconformational isomerization of prion protein, PrP, which represents thebest known process involving conformational shift of a polypeptide(PrP^(C) to PrP^(Sc)), occurs after anchorage of the PrP^(C) to theplasma membrane during internalization through the endosomal pathway,and is suspected to be assisted by a chaperone-like protein (Protein X).N terminal trimming of PrP^(Sc) to produce PrP 27-30, the material foundin amyloid deposits of infected cultured cells, occurs in secondarylysosomes, suggesting that this cell compartment is critically involvedin the pathogenesis prion diseases (Prusiner, S. B. (2001) N England JMed 344, 1516-1526).

Our data suggest that the α3(IV) NC1 conformers are secreted and undergoproteolysis via endosomal/lysosomal pathways, and that specificmodulators of GPBP regulate α3(IV)NC1 conformer production in culturedcells. All of the above suggests that α3(IV)NC1 and PrP share secretoryand degradation pathways, and, therefore GPBP may catalyze similarconformational isomerization reactions on PrP.

Fourth generation branched polyamines DAB-Am-32 have been reported toaccumulate in lysosomes and to cure prion infected cells. DAB-Am-32strongly inhibited GPBP in vitro at concentrations at which DAB-Am-4caused induction. This suggests that, as for para-amyloid matterformation, amyloid matter deposition requires GPBP action and alsosuggests that one of the mechanisms by which DAB-Am-32 eliminatesamyloid matter in prion infected cells involves inhibition of GPBP atthe lysosomal compartment. This results point to GPBP as a potentialcandidate for Protein X activity.

The results above suggest that the reported curative effect of branchedpolyamines on prion infected cells may be due in part to inhibition ofotherwise active lysosomal GPBP, thus implicating GPBP as a therapeutictarget in prion-mediated disorders.

GPBP binds to PrP^(C) in vitro. To show the biological relationshipbetween GPBP and proteins that promote amyloid matter formation whenthey undergo conformational degeneration, the interaction between GPBPand PrP^(C) was assessed in far Western assays using cellular extractsof primary cultures of rat cerebellar neurons and recombinant humanGPBP. GPBP bound to a limited number of polypeptides of different sizesall of which were recognized by specific antibodies against PrP^(C)(Santa Cruz Biotech Ca# SC7693) (FIG. 10). The presence of GPBP in thesecells was further demonstrated by Western blot analysis of thecorresponding cell extracts using specific antibodies. Furthermore, weused recombinant material, representing human GPBP and bovine PrP^(C) inspecific far Western and phosphorylation studies and found that GPBPinteracts with PrP^(C) and when incubated in the presence of [γ³²P]-ATPtransferred phosphates to PrP as a result of this interaction (FIG. 10).

Human GPBP aggregates with bovine PrP^(C). To explore further thepathway of complex formation between PrP^(C) and GPBP, we usedspectroscopy methods. Light scattering at 90° was measured for theaggregation kinetic assays. Upon addition of GPBP at a PrP^(C) solution,aggregation occurred and could be monitored by light scattering. Thecomplex formation is independent of the time course of protein additionsince the same increase in the light scattering signal is obtained whena PrP^(C) solution is added to a GPBP solution initially placed in themeasurement cell. To ascertain whether the different versions ofinhibitory Q₂ peptide (L-amino acid and D-amino acid versions of Q₂peptide —Q_(2L), Q_(2D) respectively—and Q_(2L), an inactive scrambledpeptide with the same amino acid composition than Q_(2L)) could affectGPBP-PrP^(C) complex formation in a similar manner than they affectedkinase activity, we monitored aggregation in the presence of eachindividual peptide. Upon addition of GPBP to a PrP^(C) solutioncontaining 100 μM of Q_(2L), GPBP-PrP^(C) complex formation wasefficiently inhibited. The inactive peptide Q_(2Lr) had no effect oncomplex formation at these concentrations, whereas the more potentQ_(2D) peptide at 20 μM fully inhibited GPBP-PrP^(C) complex formation.

Aggregation of GPBP and PrP^(C) depends on structural requirements forProtein X interaction. Interaction of PrP^(C) and Protein X is expectedto occur through a defined number of residues at the C terminal regionof PrP which comprises the Protein X binding site (Kaneko, K, et al.,(1997) Proc. Natl. Acad. Sci. USA 94, 10069-10074). We have performedrecombinant expression and immunoprecipitation studies in an attempt tofirst assess whether GPBP-PrP complex formation is mediated by a ProteinX-type interaction. We have used specific antibodies recognizingFLAG-tag sequence only present in recombinant GPBP to precipitateco-expressed recombinant human PrP. FLAG-specific antibodies efficientlyprecipitated FLAG-GPBP along with PrP, suggesting that FLAGantibodies-GPBP-PrP form a precipitable ternary complex and that GPBPand PrP interact in the cellular environment. When immunoprecipitationswere done on cell extracts expressing GPBP and individual PrP mutants weobserved that mutants expected to alter Protein X binding site wereprecipitated by FLAG-antibodies in a much less efficiency than mutantsnot involving these residues, which showed a similar capacity to undergoprecipitation than PrP representing wild type sequence. In FIG. 11 weillustrate a comparative study between PrP and PrP^(E168R), a human PrPmutant in which a residue proposed to be part of Protein X epitope, E68,has been replaced by R to generate a PrP^(C) mutant non-susceptible forPrP^(Sc) conversion (Kaneko, K, et al., (1997) Proc. Natl. Acad. Sci.USA 94, 10069-10074). Similar results were obtained with functionallyhomologous PrP^(Q172R) and PrP^(E219K) mutants but not with twoindependent non-functionally related PrP^(R220A) and PrP^(R228A)mutants.

GPBP promotes conformational changes in PrP. A widely used method tomonitor conformational alterations in PrP^(C) relevant to pathogenesiscomprises determination of the number of related polypeptides beingexpressed by the cell and their soluble or precipitable condition. Ingeneral, PrP^(C) is expressed inside the cell as a highly soluble singlepolypeptide and an increased number of polypeptides with poor solubilityis characteristic of PrP^(Sc) and other non-physiological conformationalforms of PrP such as PrP^(Res) or PrP^(Sc-like), and more recent datasuggest that inside the cell insoluble conformers of PrP arecontinuously being produced and cleared and that the levels of theseconformers at the steady state reflects the dynamics of these twoopposite processes (Ma, J. and Lindquist (2002) Science 298, 1785-1788).We have used inhibitors and activators of GPBP to regulate the levels ofprecipitable recombinant human PrP polypeptides in cultured cells (FIG.12). The presence of DAB-Am-4 in the culture media of cell expressingrecombinant PrP efficiently induced the expression of non-solubleprecipitable PrP polypeptides, whereas the presence of DAB-Am-32efficiently inhibited expression of non-soluble precipitable PrPpolypeptides. Similarly, we have generated and used a number of mRNAsilencers to regulate the level of expression of non-soluble PrPpolypeptides and found that individual mRNA silencers down-regulatednon-soluble PrP expression to an extent consistent with the capacitydisplayed by each individual mRNA silencer to impair endogenousnon-canonical expression of GPBP. In FIG. 13 we illustrate a comparativestudy using a non-relevant silencer (C) and two specific silencers (1,2)with higher (1) or lower (2) capacity to reduce 91- and 120-kDa GPBPexpression (see FIG. 4, lanes 5 and 7, respectively).

GPBP Bind to Aβ₁₋₄₂

Several lines of evidence suggest that senile plaques in Alzheimer'sdisease derive from neurons that have undergone degeneration primarilycaused by amyloid deposition of Aβ₁₋₄₂ at secondary lysosomes (Nixon, etal., (2000) Neurochem Res 25, 1161-1172; Andrea, M R, et al (2001) 38,120-134). Conceivably, a similar mechanism to that proposed above to bemediating amyloid matter formation in prion diseases could be mediatingamyloid matter deposition in Alzheimer's disease. To assess thispossibility the capacity of GPBP to bind to Aβ₁₋₄₂ was assessed inspecific far Western studies (FIG. 14). Recombinant human GPBP displayedhigh affinity for a synthetic polypeptide representing Aβ₁₋₄₂, whereasin the same assay conditions GPBP did not display binding capacitytowards a synthetic peptide representing the non-phosphorylable Nterminal region of bovine α3(IV)NC1. Incubation of GPBP with Aβ₁₋₄₂ inthe presence of [γ³²P]ATP did not result in ³²P-labeling of syntheticpolypeptide, suggesting that although Aβ₁₋₄₂ contains sites for GPBPmolecular recognition, it does not harbor GPBP phosphorylation sites.Consequently, Aβ₁₋₄₂ perhaps represents a substrate of GPBP for aconformational catalysis in which phosphate transfer of proteinsubstrate is not required. The latter suggests that GPBP-mediatedconformational catalysis on protein substrates can occur in aphosphorylation-dependent or independent manner, or that conformationalcatalysis can be performed on phosphorylated or non-phosphorylatedsubstrates. Consistently, GPBP bound with more affinity to recombinantproteins representing phosphorylated version of human autoantigens(Goodpasture antigen and myelin basic protein) at specific Ser thatconform phosphorylation sites for GPBP (Ser⁹ and Ser⁸, respectively),suggesting that the phosphorylated products are not the end product ofGPBP catalysis, but they are the substrate for a conformationalisomerization and supramolecular assembly catalysis.

In full our data provide the first experimental support for GPBP beingthe chaperone-like molecular enzyme suspected to be involved in PrP^(C)to PrP^(Sc) conformational isomerization in the pathogenesis of priondiseases, and also represents the first molecular link between twopreviously unrelated processes, tissue degeneration mediated by amyloidand para-amyloid matter deposition and autoimmunity.

DISCUSSION

Autoimmune diseases comprise a large number of disorders mediated by animmune attack against self-components (autoantigens) as a result of afailure in the mechanisms of immune tolerance. When autoantigens areadministered to animal models they have the peculiar capacity to engagethe immune system in a response that mimics the natural diseaserevealing that these components display biological features ofimmunological relevance. Consequently, certain alterations inautoantigen biology could have an important impact in theirimmunological recognition, thus triggering an immune response. For thesereasons, understanding autoantigen biology is necessary to design anappropriate molecular model for autoimmune disorders.

By studying Goodpasture (GP) disease we have provided new insights intothe molecular mechanism of autoimmune disorders. GP disease ischaracterized by the coexistence of glomerulonephritis and lunghemorrhage caused by an immune attack that is mediated by circulatingautoantibodies, which deposit in a linear manner in the glomerular andalveolar basement membranes. The autoantibodies are directed against theC terminal non-collagenous domain (NC1) of the α3 chain of collagen IV,α3(IV)NC1 domain, also called the Goodpasture antigen. Collagen IV iscomposed of six α-chains that exhibit a high degree of homology which ismore evident at the NC1 domain. However, only the α3(IV)NC1 domain hasbeen shown to induce Goodpasture syndrome in animals models, and onlythe human α3(IV)NC1 domain has been implicated in a common naturalautoimmune response. Comparative structural studies identified a highlydivergent region at the N terminus of the α3(IV)NC1 domain whichundergoes phosphorylation by cAMP-dependent protein kinase and also byGPBP. α3(IV)NC1 domain is purified from natural sources as a set ofconformational isomers (conformers) with differential phosphoserinecontent. The more abundant α3(IV)NC1 conformer, which likely representsthe native conformation, is virtually devoid of phosphoserine, whereasthe less abundant α3(IV)NC1 conformers, likely representing derivedalternative conformations, display the highest degree of phosphoserinecontent. These data suggested that phosphorylation is part of thestrategy used by cells to generated alternative protein conformations(WO 00/50607; WO 02/061430).

Other biological consequences associated with phosphorylation of theα3(IV)NC1 domain include regulation of α3(IV)NC1 domain aggregation. Inthe absence of ATP, GPBP displays the capacity to catalyze aphysiological aggregation of the α3(IV)NC1 domain (disulfide-dependentoligomerization) by a process involving conformational isomerization (WO02/061430). This data indicate that GPBP possesses a conformationalisomerase activity independent of its kinase activity that is criticalfor a broader enzymatic catalysis, resulting in assembly of a proteinsubstrate into a quaternary structure.

The relationship between GPBP and autoimmune pathogenesis was initiallyestablished by showing (a) elevated levels of GPBP in Goodpasturepatients; (b) In vitro, GPBP catalyzes the production of α3(IV)NC1conformers that are found in patient kidneys but not control kidneys;and (c) The presence of aberrant (α3(IV)NC1 conformers in patientkidneys that are specifically recognized by pathogenic autoantibodies.(WO 02/061430) These results led to a new model of autoimmune disease,wherein the autoimmune response is considered a legitimate reaction ofthe immune system against aberrant conformations of an autoantigen (suchas the α3(IV)NC1 in GP disease), which assemble and for which the immunesystem has not established a tolerance (WO 00/50607; WO 02/061430).

These observations identify critical and exclusive biological featuresin a human autoantigen (the α3(IV)NC1) that do not have a counterpart inhomologous domains (the other type IV collagen NC1 domains) which arenot autoantigens, and represent a new strategy to study the molecularbasis of a human exclusive disease. Recent studies performed in ourlaboratory have identified the presence of a serine residue in the Nterminal region of myelin basic protein (“MBP”) that is structurally andfunctionally similar to that found in the α3(IV)NC1 (WO 02/061430).Myelin basic protein is a major autoantigen in an autoimmune responsemediating multiple sclerosis, which, like Goodpasture disease, is anexclusively human autoimmune disease. Recent studies furtherdemonstrated that phosphorylation of MBP plays a critical role inregulating its conformation and have identified conformational-dependentdifferences in the proteolytical susceptibility of myelin basic proteinfrom control and patients affected by multiple sclerosis (data notshown). These data represent a strong validation of our model ofautoimmune disease, based on the biology of two unrelated humanautoantigens and autoimmune disorders, and suggest that a commonautoimmune pathogenic mechanism is emerging in which GPBP plays acentral role. In this mechanism, human autoantigens are polypeptideswith the capacity to bind to GPBP and, as a consequence of such abinding, undergo phosphorylation and conformational isomerization, whichmakes these polypeptides vulnerable to an aberrant catalysis andproduction of non-tolerized conformers (WO 00/50607; WO 02/061430).

The high phosphorylability of myelin basic protein in vitro contrastswith its low content in Ser(P) residues, suggesting that (a) endogenousphosphorylation of this autoantigen is highly regulated; and (b) thereare multiple MBP species with different degree of phosphorylation[Eichberg, J., & S. Iyer, Neurochem Res 21, 527-535 (1996)].Conceivably, the sequential phosphorylation and dephosphorylation ofspecific sites on autoantigens, such as those identified at the Nterminus in myelin basic protein and in the human α3(IV)NC1 domain,could generate a heterogenous population of molecules or conformers forsupramolecular assembly, and an alteration in the homeostasis ofphosphorylation events could result in the assembly of aberrantnon-tolerized conformers in the corresponding quaternary structure ofthe autoantigens.

GPBP displays a number of biological features to be considered a goodcandidate as a pivotal component of the cellular machinery catalyzingthe supramolecular assembly of autoantigens and inducing immune responseduring autoimmune pathogenesis. For example: (1) GPBP phosphorylateshomologous sites in two different human autoantigens and targets otherhuman autoantigens; (2) The GPBP phosphorylation sites in myelin basicprotein and Goodpasture antigen play a conformational regulatory role;(3) GPBP binds preferentially to recombinant species representing thephosphorylated versions at these sites, suggesting that thephosphorylated versions are not only the product of a phosphate transferreaction, but are also the substrate of an additional catalysis thatincludes conformational isomerization and supramolecular assembly; (4)Immunochemical studies show that GPBP is present in tissue, cellular andsubcellular localizations that are common targets of autoimmuneresponses; (5) Increased levels of GPBP relative to its alternativelyspliced isoform, GPBPΔ26, are found in several autoimmune conditions (WO00/50607; WO 02/061430 and data not shown).

To further establish the role of GPBP in autoimmune pathogenesis, amajor issue is to determine the mechanism by which GPBP is delivered tosuch a broad number of subcellular localizations. Proteins can besynthesized at free ribosomes (proteins to be resident at the cytosol orto be further transported to, for example, nucleus, mitochondria orperoxisome) or at ribosomes associated with ER (proteins that enter intothe secretory pathway and end up being either ER, Golgi apparatus,lysosomes and plasma membrane resident, or secreted to the extracellularmatrix). There are proteases present in all these locations, and thereare many examples in which primary translation products undergoproteolysis to render shorter biologically active polypeptides.

In the cell, protein sorting is accomplished via a number of signalsequences, many of which have been characterized. However, there areincreasing examples of non-canonical mechanisms for cellular proteinsorting.

By studying the cellular expression of GPBP, we have established thatthe cell expresses at least seven GPBP-related polypeptides of 120-,91-, 77-, 60-, 44-47-, 32-kDa. With the exception of 120-kDa GPBP, whoseorigin is not certain, the rest can be generated by limited proteolysisof the 91-kDa polypeptide, as shown herein. We present evidencesuggesting that the 91 kDa GPBP is a non-canonical translation productof GPBP mRNA. The evidence presented herein also suggests that 91 kDaGPBP enters into the secretory pathway and undergoes processing toproduce GPBP isoforms of lower molecular mass that can be found in theER, Golgi apparatus, lysosomes and plasma membrane.

Confocal studies performed in our laboratory show a majorco-localization of GPBP and Goodpasture antigen in human glomerulussuggesting the presence of GPBP in basement membranes. In contrast, ourevidence from recombinant expression studies suggests that canonical77-kDa polypeptide is essentially cytosolic (data not shown). However,subfractioning studies show that at the cellular steady state the levelsof canonical primary product are negligible and only a major derivedproduct of 60-kDa can be detected, suggesting that the 77-kDa primaryproduct, if it is expressed, undergoes an efficient processing to alower molecular mass isoform. The mechanisms for GPBP transport to thenucleus and mitochondria (WO 00/50607; WO 02/061430) remain to beverified, although our data suggests that certain non-canonicaltranslation products may provide the requisite targeting signals forsuch localization.

Recombinant expression shows that the 5′-UTR contains multiplenon-canonical sites for translation initiation that display a 5′ to 3′hierarchy. Based on sequence analysis and using programs that predictsubcellular localization, the ORF in Δ102 contains a canonical signalpeptide sequence to entry into the secretory pathway (residues 1-46).This signal peptide is immediately followed by a signal for nuclearlocalization (residues 47-50) and another for mitochondrial destination(residues 52-56), in turn, suggesting that by varying transcriptioninitiation site the cell may regulate the expression of non-canonicalpolypeptides that are destined for the secretory pathway (ER/Golgiapparatus/lysosomes/plasma membrane/extracellular matrix), nuclear omitochondrial whereas only canonical translation would generate agenuine cytosolic polypeptide. Furthermore, GPBP also displays two otherpotential mechanism to reach nuclear environment: (a) GPBP contains abipartite nuclear localization signal; and (b) GPBP binds to a family oftranscription factors that could shuttle the protein into the nucleus(WO 03/048193).

The cellular expression of GPBPΔ26 was also explored using Mab14, amonoclonal antibody recognizing both GPBP and GPBPΔ26 recombinantcounterparts. Mab14 reacted with a single 77-kDa cytosolic polypeptideand did not show significant reactivity towards polypeptides reactingwith Mab6. The specificity of these Mab14 antibodies was confirmed bydemonstrating that GPBP/GPBPΔ26 silencers reduced the expression of77-kDa polypeptide to similar extent than 91- and 120-kDa polypeptidesthat only reacted with Mab6. These results suggest that the 77-kDapolypeptide is primarily GPBPΔ26 and cytosolic, whereas non-canonicalpolypeptides are mainly GPBP, and virtually ubiquitous.

In summary, our data suggest that for native cellular expression Mab14is an immunological probe for GPBPΔ26 whereas Mab6 is an immunologicalprobe for GPBP-related polypeptides.

Our findings suggest that GPBP is an integral component of theendosomal-lysosomal pathway which activity is regulated in part by acatepsin-dependent processing, a biological strategy described for otherenzymes (Pham, C. T., & T. J. Ley, (1999). Proc Natl Acad Sci U S A96(15): 8627-8632). These proteases are critical in processing proteinsentering the endosomal pathway, and for producing peptides that arepresented through MHC class II (Chapman, H. A., (1998) Curr Opin Immunol10(1): 93-102). Disturbances of lysosomal environment in a generalmanner, such as modifying the pH using compounds as chloroquine, or in aspecific manner using catepsin inhibitors such as leupeptin, have beenshown to alter peptide presentation by MHC class II (Demotz, S., P. M.Matricardi, C. Irle, P. Panina, A. Lanzavecchia, & G. Corradin, (1989) JImmunol 143(12): 3881-3886; Turk, V., B. Turk, & D. Turk, (2001) EMBO J20(17): 4629-4633). We have shown herein that leupeptin treatmentsubstantially alters lysosomal processing of GPBP and therefore alsolikely induces an alteration in GPBP activity, which in turn suggeststhat altered peptide presentation and altered GPBP activity may berelated and perhaps critical in autoimmune pathogenesis, whichnecessarily requires aberrant peptide presentation to be effective.

A feature common to many degenerative diseases is the formation ofdeposits of specific polypeptides. Where and how these deposits appearis highly specific and tightly related with pathogenesis. The depositscan be nuclear inclusion bodies, as in cerebellar ataxia, or be at theER lumen, such as in some degenerative disease affecting liver andneurons, or be cytoplasmic inclusion bodies, as in Parkinson's disease,Alzheimer's disease, and amyotrophic lateral sclerosis; andendosomal-lysosomal, as in Alzheimer's disease, prion diseases, and typeII diabetes. GPBP is an ubiquitous protein that has been independentlyrelated to conformational catalysis of substrate proteins (WO 00/50607;WO 02/061430) and in the formation of protein deposits in animal modelsthat develop a degenerative nephropaty associated to an autoimmuneresponse. Consequently the finding that GPBP interacts with PrP andAβ₁₋₄₂ two polypeptides that undergo conformational alteration and formamyloid deposits in prion and Alzheimer's disease, respectively,represents strong evidence for GPBP being involved in the pathogenesisof these degenerative disorders. More specifically a protein resident inthe endosomal-lysosomal pathway named Protein X has been proposed tobind to PrP and catalyze the conformational transition from PrP^(C) toPrp^(Sc) (Prusiner, S. B., (1998). “Prions.” Proc Natl Acad Sci USA95(23): 13363-13383.). Here we present evidence indicating that GPBPbinds to PrP in a Protein X fashion, phosphorylates PrP, formsaggregates with it and, as a consequence of this interaction, PrPundergoes conformational changes that renders PrP highly insoluble andprecipitable. To our knowledge, GPBP represents the best molecularcandidate to be Protein X in prion diseases as well as to perform asimilar catalytical role in other protein deposit-mediated humandisease.

A major obstacle when studying the molecular basis of degenerative orautoimmune diseases is the almost general consensus that any protein canbe an autoantigen or to conformationally degenerate and form deposits.According to this view, the establishment of an autoimmune responserepresents a non-legitimate immune reaction, while conformationaldegeneration is thought to represent a stage that any polypeptide chaincan achieve if the environment is appropriately altered. However, thisview cannot explain the principal fact that only a very limited numberof cellular components can be autoantigenic or can form deposits thatcause tissue degeneration, indicating that autoantigens anddeposit-forming polypeptides share biological features. Our studiessuggest that a common biological feature of autoantigens is being asubstrate of an enzymatic strategy to form quaternary structures inwhich GPBP plays a central role and the protein substrate undergoesconformational isomerization. Our results regarding polypeptides that,like PrP and Aβ₁₋₄₂, conformationally degenerate and form deposits,suggest that they are also substrates of GPBP and its catalytic actionis required for deposit formation. While the present invention is notlimited to a specific mechanism, we propose that GPBP is a novelmolecular enzyme that binds to and phosphorylates protein substrates aspart of an enzymatic strategy in which conformational catalysis ofprotein substrates occur during their supramolecular assembly(quaternary structure). Alterations in its performance produce aberrantconformers that are soluble and induce autoimmunity, or are insolubleand form deposits of amyloid or para-amyloid nature that cause tissuedegeneration.

1.-27. (canceled)
 28. A method for treating a an autoimmune conditioncomprising administering to a subject in need thereof an amounteffective to treat the disorder of a polypeptide comprisingX1-SHCIX2-X3; wherein X1 is 0-10 amino acids of the sequence ATTAGILATL(SEQ ID NO:41); X2 is E or Q; and X2 is 0-10 amino acids of the sequenceLMVKREDSWQ (SEQ ID NO:42). 29.-33. (canceled)
 34. The method of claim 28wherein X2 is E.
 35. The method of claim 28 wherein X2 is Q.
 36. Themethod of claim 28 wherein X1 is ILATL.
 37. The method of claim 28wherein X3 is LMVKR.
 38. The method of claim 28, wherein the polypeptidecomprises LATLSHCIELMVKR (SEQ ID NO:90).
 39. The method of claim 28,wherein the polypeptide consists of LATLSHCIELMVKR (SEQ ID NO:90). 40.The method of claim 39, wherein the polypeptide comprises D amino acids.41. The method of claim 28 wherein the autoimmune condition is selectedfrom the group consisting of Goodpasture Syndrome, multiple sclerosis,systemic lupus erythematosus, cutaneous lupus erythematosus, pemphigus,pemphigoid and lichen planus.